Imitation SWI
ATP-dependent chromatin
remodeling in vitro was first discovered in transcription factor-mediated disruption of nucleosome arrays. Previously identified SWI/SNF complexes were then shown to be
ATP-dependent chromatin-remodeling factors (Cote, 1994; Imbalzano, 1994; Kwon,
1994; Wang, 1996a and b). Subsequent studies identified NURF (nucleosome remodeling factor)
(Tsukiyama, 1995), RSC (remodeling structure of chromatin) (Cairns, 1996), CHRAC
(chromatin accessibility complex) (Varga-Weisz, 1997), ACF (ATP-utilizing chromatin assembly
and remodeling factor) (Ito, 1997), Mi-2 complex (Wade, 1998; Zhang, 1998), and
RSF (remodeling and spacing factor) (LeRoy, 1998) as ATP-dependent chromatin-remodeling
complexes. Interestingly, all of the ATP-dependent chromatin-remodeling factors identified thus far have
the putative ATPase subunit that belongs to the SWI2/SNF2 superfamily (Eisen, 1995). NURF
(Tsukiyama, 1995), CHRAC (Varga-Weisz, 1997), and ACF (Ito, 1997) share
Drosophila ISWI (Imitation Switch) protein (Elfring, 1994) as a putative ATPase subunit. The
Drosophila ISWI gene was originally cloned based on its homology within the ATPase domain to the
Brahma gene, the SWI2/SNF2 homolog in flies. Phylogenetic studies reveal that
the ISWI genes are highly conserved during evolution and form a distinct subfamily within the
SWI2/SNF2 superfamily (Eisen, 1995). This suggests that the ISWI genes have indispensable
functions in vivo.
NURF facilitates transcription factor-mediated disruption of physiologically spaced nucleosomal arrays.
This leads to the establishment of accessible chromatin structure at the promoter regions of genes
(Tsukiyama, 1995) and facilitation of transcription from chromatin templates in vitro (Mizuguchi, 1997). CHRAC increases the access of restriction enzymes to chromatin DNA and facilitates
regular spacing of nucleosomes (Varga-Weisz, 1997) as well as T antigen-dependent replication of
SV40 DNA in vitro (Alexiadis, 1998). ACF facilitates both regular spacing of nucleosome arrays
and transcription factor-mediated disruption of nucleosome arrays in the presence of histone chaperones (Ito, 1997).
It is interesting that there are two copies each of genes that belong to the SWI2/SNF2 and ISWI
subfamilies in both humans and yeast (human SWI2/SNF2, hbrm and BGR-1; human
ISWI, hSNF2L and hSNF2h; yeast SWI2/SNF2, SWI2/SNF2 and STH1; yeast ISWI, ISW1 and ISW2). In
mammalian systems, biochemical activities of BRG1 and hbrm complexes are indistinguishable. However, BRG1 and hbrm are differentially regulated on
mitogen-induction of cell growth and ras-mediated oncogenic transformation of cells, demonstrating that they may have distinct sets of target genes. Yeast complexes containing
SWI2/SNF2p and STH1p have similar biochemical activities but
have clearly distinct functions in vivo, because SWI2/SNF2 is nonessential whereas STH1 is essential. ISW1 and ISW2 complexes have distinct biochemical
activities, suggesting that they may have distinct functions in vivo. These data indicate that multiple
ATP-dependent chromatin remodeling complexes with distinct functions are required in both lower and higher eukaryotes (Tsukiyama, 1999 and references).
Two Saccharomyces cerevisiae Imitation Switch genes (ISW1 and ISW2) have been identified and characterized. They code for proteins that are highly related to
Drosophila ISWI, encoding the putative ATPase subunit of three ATP-dependent chromatin remodeling factors. Purification of ISW1p reveals a
four-subunit complex with nucleosome-stimulated ATPase activity, as well as ATP-dependent nucleosome disruption and spacing activities.
Purification of ISW2p reveals a two-subunit complex also with nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing
activities but no detectable nucleosome disruption activity. Null mutations of ISW1, ISW2, and CHD1 (see Drosophila Chd1) genes cause synthetic lethality in various
stress conditions in yeast cells, revealing the first in vivo functions of the ISWI subfamily of chromatin-remodeling complexes and
demonstrating their genetic interactions. A single point mutation within the ATPase domain of both ISW1p and ISW2p inactivates all
ATP-dependent biochemical activities of the complexes, as well as the ability of the genes to rescue the mutant phenotypes. This demonstrates
that the ATP-dependent chromatin-remodeling activities are essential for the in vivo functions of both ISW1 and ISW2 complexes (Tsukiyama, 1999).
This paper presents a molecular genetic characterization of ISW2, identified in a screen of a yeast lambdagt11 library using a monoclonal antibody that reacts with a 210 kDa mammalian microtubule-interacting protein. The protein is 50% identical to the Drosophila nucleosome remodelling factor ISWI and is therefore a new member of the SNF2 protein family. Although not essential for vegetative growth, the ISW2 gene is required for early stages in sporulation. The isw2 homozygous deletant diploid strain is blocked in the G(1) phase of the cell cycle, unable to execute the premeiotic DNA replication and progress through the nuclear meiotic division cycle. ISW2 expression from a multicopy plasmid has the same effect as deletion, but ISW2 expression from a centromeric plasmid rescues the deletion phenotype. In vegetatively growing diploid cells, the Isw2 protein is preferentially found in the cytoplasm, co-localizing with microtubules. An accumulation of the Isw2 protein within the nucleus is observed in cells entering sporulation. A role is proposed for the Isw2 protein in facilitating chromatin accessibility for transcriptional factor(s) that positively regulate meiosis/sporulation-specific genes (Trachtulkova, 2000).
The yeast Isw2 chromatin remodeling complex functions in parallel with the
Sin3-Rpd3 histone deacetylase complex to repress early meiotic genes upon
recruitment by Ume6p. For many of these genes, the effect of an isw2 mutation is partially masked by a functional Sin3-Rpd3 complex. To identify the full range of genes repressed or activated by these factors and uncover hidden targets of Isw2-dependent regulation, full genome expression analyses were performed using cDNA microarrays. The Isw2 complex was found to function mainly in repression of transcription in a parallel pathway with the Sin3-Rpd3 complex. In addition to Ume6 target genes, many Ume6-independent genes are derepressed
in mutants lacking functional Isw2 and Sin3-Rpd3 complexes. Conversely, ume6 mutants, but not isw2 sin3 or isw2 rpd3 double mutants, have reduced fidelity of mitotic chromosome segregation, suggesting that one or more functions of Ume6p are independent of Sin3-Rpd3 and Isw2 complexes. Chromatin structure analyses of two nonmeiotic genes reveal increased DNase I sensitivity within their regulatory regions in an isw2 mutant. These data suggest that the Isw2 complex functions at Ume6-dependent and -independent loci to create DNase I-inaccessible chromatin structure by regulating the positioning or placement of nucleosomes (Fazzio, 2001).
The ISWI class of chromatin remodeling factors exhibits potent chromatin
remodeling activities in vitro. However, the in vivo functions of this class of
factors are unknown at a molecular level. S. cerevisiae Isw2
complex represses transcription of early meiotic genes during mitotic growth in
a parallel pathway to Rpd3-Sin3 histone deacetylase complex. This repressor
function of lsw2 complex is largely dependent upon Ume6p, which recruits the
complex to target genes. Nuclease digestion analyses reveal that lsw2 complex
establishes nuclease-inaccessible chromatin structure near the Ume6p binding
site in vivo. Based on these findings, a model is proposed for the mechanism of
transcriptional repression by two distinct chromatin remodeling complexes (Goldmark, 2000).
To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, a system to assemble nucleosomal arrays on immobilized templates has been developed using recombinant yeast core histones. This system enabled analysis of the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal
arrays. Isw2 complex was found to interact efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction. The second subunit of Isw2
complex, encoded by open reading frame YGL 133w (herein named ITC1), was identified and both subunits of the complex, Isw2p and Itc1p, were found to be essential for efficient interaction with DNA and nucleosomal arrays. Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex. Finally, it was found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes. These results demonstrate the utility of the in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays (Gelbart, 2001).
Activity of monomeric Drosophila ISWI protein was compared with that of sw2p and Itc1p subunits. The requirement of both Isw2p and Itc1p
subunits in all assays (DNA and nucleosome binding, ATPase, and chromatin remodeling) was unexpected. The ATPase assay was performed using 0.2 pmol of Isw2p and nucleosomes containing 25 ng of DNA in a 5-µl reaction, a condition identical to that in the study of Drosophila ISWI. Even under this condition, nucleosome-stimulated ATPase activity of monomeric Isw2p could not be detected. This result implies that Isw2p may be different from Drosophila ISWI protein and lacks biochemical activities as a monomer. However, it
should be noted that monomeric Drosophila ISWI protein is extremely labile, and its biochemical activities can easily be lost upon freeze-thaw cycles or
prolonged storage. This implies that minor differences in the folding properties of monomeric Drosophila ISWI protein and Isw2p may account for the observed differences in their biochemical activities. It is also possible that biochemically active monomer Isw2p needs to be purified under special conditions yet to be determined. While active in some biochemical assays, Drosophila ISWI exhibits significantly higher specific activities when incorporated into complexes according to two reports. One study shows that three to five fmol of NURF and 0.14 to 0.28 pmol of monomeric ISWI exhibit comparable ATPase activities. Additionally, another study shows that 2.2 but not 0.22 pmol of monomeric Drosophila ISWI is active in the nucleosome spacing assay, and 22 fmol of recombinant ACF exhibits a comparable activity. In contrast, Drosophila CHRAC and E. coli-expressed monomeric ISWI exhibit comparable specific activities in ATPase, nucleosome spacing, and nucleosome disruption assays. The
basis for the differences among these reports remains unknown (Gelbart, 2001 and references therein).
The packaging of the eukaryotic genome in chromatin presents barriers that
restrict the access of enzymes that process DNA. To overcome
these barriers, cells possess a number of multi-protein, ATP-dependent chromatin
remodelling complexes, each containing an ATPase subunit from the SNF2/SWI2
superfamily. Chromatin remodelling complexes function by
increasing nucleosome mobility and are clearly implicated in transcription. SNF2/SWI2- and ISWI-related proteins have been analyzed
to identify remodelling complexes that potentially assist other DNA transactions.
A complex has been purified from Saccharomyces cerevisiae that contains the
Ino80 ATPase. The INO80 complex contains about 12 polypeptides
including two proteins related to the bacterial RuvB DNA helicase,
which catalyses branch migration of Holliday junctions. The purified complex
remodels chromatin, facilitates transcription in vitro and displays
3' to 5' DNA helicase activity. Mutants of ino80 show hypersensitivity
to agents that cause DNA damage, in addition to defects in transcription. These results indicate that chromatin remodelling driven by the
Ino80 ATPase may be connected to transcription as well as DNA damage repair (Shen, 2000).
The Saccharomyces genome database was searched for genes that are
highly related to Drosophila ISWI, which codes for the ATPase
subunit of the nucleosome remodelling factor NURF.
SWI2/SNF2, STH1, ISWI, ISW2, CHD1 and the
three open reading frames (ORFs) YFR038W, YGL150C and
YDR334W were found. YGL150C was identified
in a genetic screen for mutants affecting inositol biosynthesis. Previously
it had been called ARI1 (for ATPase Related to ISWI); hereafter,
INO80 will be used. Typical of SNF2/SWI2 family proteins, the conserved ATPase
domain of Ino80 comprises a large proportion of the coding region. Beyond
the ATPase domain, Ino80 does not possess the SANT domain found in Isw1 and
Isw2; however, sequence comparison
of INO80 and its counterparts from human (hINO80) and Drosophila
(dINO80) reveals two additional conserved regions: the TELY motif
at the amino terminus and the GTIE motif at the carboxy terminus (Shen, 2000).
Most, if not all, of Ino80 is present in a large complex. The complex of proteins associated with Ino80, the 'INO80 complex' (INO80.com), was purified by immunoprecipitation. The purified INO80 complex contains 12 principal polypeptides, most of which, with notable exceptions, are present at roughly equivalent stoichiometry. Actin (Act1) and three actin-related proteins, Arp4, Arp5 and Arp8, are associated with the complex in addition to Ino80. beta-Actin is a functional component of the mammalian BAF complex, and Arp7 and Arp9 are shared by the yeast SWI/SNF and RSC complexes. Actin and Arp4 are
also contained in the yeast NuA4 histone acetyltransferase complex.
Two other intensely staining polypeptides of the INO80 complex, Rvb1 and Rvb2,
are encoded by the ORFs YDR190C and YPL235W, respectively. Rvb1
and Rvb2 were previously identified as 'RuvB-like' proteins
(scRUVBL1/scTIP49a and scRUVBL2/scTIP49b) sharing homology to bacterial RuvB, the Holliday junction DNA helicase; both RVB1 and RVB2 genes are essential in yeast. The first RuvB-like protein (TIP49/rTIP49a) was identified in rat, and a pair of RuvB-like proteins (hTIP49a/RUVBL1/TIP49 and hTIP49b/TIP48)
are found in human and are associated in large complexes (Shen, 2000).
Like other ATP-driven chromatin remodelling complexes, the INO80 complex
has ATPase activity. The ATPase
activity is intrinsically high, however, and is not further stimulated by
exogenous DNA. The
bulk of this ATPase activity is ascribed to Ino80. The inability to stimulate
ATPase activity by exogenous DNA might be caused by the presence of endogenous
DNA associated with the INO80 complex. DNA of heterogeneous size was detected
in the purified preparation. DNase I digestion has no observable
effects on the polypeptide composition of the INO80 complex, but the intrinsic ATPase activity of the INO80
complex is substantially reduced.
The ATPase activity of the DNase I-treated complex could then be stimulated
with exogenous DNA, or
with nucleosomes. Thus, like the related remodelling complexes
SWI/SNF and RSC, the INO80 complex shows DNA-dependent
ATPase activity (Shen, 2000).
Among the polypeptides associated with Ino80, Rvb1 and Rvb2 are of special
interest. The staining intensity of Rvb1 and Rvb2 protein bands in the INO80 complex
is unusually high in relation to other polypeptides.
The molar ratios for Rvb1 and Rvb2 relative to Act1 are 6.5 and 5.6, whereas the ratios relative to
Ino80 are 5.3 and 5.6. Thus, to a first approximation, both Rvb1 and
Rvb2 show 6:1 stoichiometry compared with other polypeptides in the INO80
complex. The correspondence between these values and the known double hexamer
composition of bacterial RuvB is striking (Shen, 2000).
Given that bacterial RuvB is an ATP-dependent DNA helicase,
an investigation was carried out to see whether the INO80 complex containing Rvb1 and Rvb2 subunits has helicase activity. The INO80 complex is able to displace a radiolabelled 52-nucleotide
DNA strand from a duplex formed by annealing the oligonucleotide to single-stranded PhiX174
DNA. Primer displacement is ATP-dependent,
as noted for other helicases such as TFIIH. In contrast, Drosophila
NURF shows no evidence of helicase activity, despite its potency in chromatin
remodelling. The Ino80-K737A mutant complex fails to show DNA helicase
activity, indicating that Rvb1 and Rvb2 may
be functionally coupled to the Ino80 ATPase.
To examine the directionality of helicase action, a substrate consisting
of 24-mer and 32-mer oligonucleotides annealed to the corresponding ends of
linear, single-stranded PhiX174 DNA was used.
The INO80 complex is able to displace only the 24-mer, indicating a 3'
to 5' helicase activity. The directional
helicase activity of the INO80 complex is also ATP dependent (Shen, 2000).
The INO80 complex might function indirectly in DNA repair by facilitating
transcription of genes induced by DNA damaging agents; however, normal induction of the ribonucleotide reductase genes RNR1 and
RNR3 (which are the downstream targets of the DNA damage checkpoint gene
MEC1 were found after treatment of ino80
cells with hydroxyurea. Alternatively, chromatin remodelling
by the INO80 complex might directly facilitate an aspect of DNA metabolism
involving replication, recombination or repair, processes that are intimately
connected. The INO80 complex might either promote site recognition
by the replication or repair machinery, or assist progression of the machinery
through chromatin. In Escherichia coli, the RuvAB complex binds to
Holliday junctions and promotes branch migration, the motive force being provided
by the helicase activity of the two hexameric RuvB rings. The RuvAB complex also has a role in the generation and prevention of double-stranded breaks at arrested replication forks through its actions on potential Holliday junction intermediates. The yeast Rvb1 and Rvb2 hexamers in
the INO80 complex might function similarly, but with the added coupling to
chromatin remodelling proteins as an adaptation to the chromatin environment.
These results, together with the recent findings of human RuvB-like proteins
being associated with c-Myc and being contained in the multisubunit
TIP60 HAT complex (see Drosophila Tip60), underscore the linkage of this class of
DNA helicases with diverse aspects of chromatin metabolism (Shen, 2000).
Tetrahymena Histone Acetyltransferase A, p55, is a homolog of yeast Gcn5p which exists as a heterotrimeric complex in yeast cells with at least two other polypeptides. Current models suggest that this complex bridges enhancer-binding factors to the basal transcription machinery. Both the Tetrahymena protein and Gcn5p possess histone acetyltransferase activity and a highly conserved bromodomain. p55 preferentially acetylates histone H3. The presence, in nuclear A-type histone acetyltransferases but not in cytoplasmic B-type HATs, of a bromodomain, known to function in protein-protein interaction, suggests that HAT A is directed to chromatin through protein interaction to facilitate transcriptional activation. It is also believed that there is a functional interaction between the Histone Acetyltransferase A type of complex and components of the SWI-SNF complex in yeast (Drosophila homologs: ISWI and Brahma). Although the exact mechanism by which the SWI/SNF complex operates is unclear, an implication of this interaction is that one function of the SWI/SNF complex is to direct HAT A to specific sites in chromatin. These findings, combined with the recent demonstration that the SWI/SNF polypeptides are integral components of the RNA polymerase II holoenzyme in yeast, suggest a mechanism whereby HAT A is targeted to chromatin during transcriptional activation, establishing a direct link between histone acetylation and gene activation (Brownell, 1996).
Isw1p and Isw2p are budding yeast homologs of the Drosophila ISWI chromatin-remodeling ATPase. Using indirect-end-label and chromatin immunoprecipitation analysis, independent and cooperative Isw1p- and Isw2p-mediated positioning of short nucleosome arrays in gene-regulatory elements is shown at a variety of transcription units in vivo. Evidence that both yeast ISWI complexes regulate developmental responses to starvation and that for Isw2p, recruitment by different DNA-binding proteins controls meiosis and haploid invasive growth (Kent, 2001).
Many studies have established that the Swi/Snf family of chromatin-remodeling complexes activate transcription. Recent reports have
suggested the possibility that these complexes can also repress transcription. Chromatin immunoprecipitation evidence is presented
that the Swi/Snf complex of Saccharomyces cerevisiae directly represses transcription of the SER3 gene. Consistent with its role in
nucleosome remodeling, Swi/Snf controls the chromatin structure of the SER3 promoter. However, in striking contrast to activation by
Swi/Snf, which requires most Swi/Snf subunits, repression by Swi/Snf at SER3 is dependent primarily on one Swi/Snf component, Snf2.
These results show distinct differences in the requirements for Swi/Snf components in transcriptional activation and repression (Martens, 2002).
The findings for Swi/Snf, taken together with recent reports that show the physical presence of RSC and Isw2 at the promoters of repressed genes, provide strong evidence for nucleosome-remodeling complexes acting
directly to repress transcription. Although Swi/Snf associates with the SER3 promoter, the mechanism of Swi/Snf recruitment to this promoter
remains unknown. Swi/Snf may be recruited through interaction with a DNA-binding protein, in a manner similar to Isw2 recruitment by the Ume6 repressor. Alternatively, Swi/Snf might have binding specificity for a particular chromatin structure at the SER3 promoter (Martens, 2002).
There are several possible mechanisms by which Swi/Snf could repress transcription. (1) Based on in vitro experiments showing that Swi/Snf can catalyze remodeling of nucleosomes in either direction between the inactive and remodeled states, Swi/Snf might create an inactive nucleosome conformation at SER3 that prevents transcription-factor access to the promoter. (2) Swi/Snf could facilitate the binding of a
transcriptional repressor of SER3. (3) Swi/Snf nucleosome remodeling could facilitate a subsequent step required for repression, such as histone modification. Recent studies have shown that some Swi/Snf-related complexes associate with histone deacetylase activity (Martens, 2002).
There are several classes of ATP-dependent chromatin remodeling complexes, which modulate the structure of chromatin to regulate a variety of cellular processes. The budding yeast, Saccharomyces cerevisiae, encodes two ATPases of the ISWI class, Isw1p and Isw2p. Isw1p copurifies with three other proteins. These associated proteins have been identified; Isw1p forms two separable complexes in vivo (designated Isw1a and Isw1b). Biochemical assays revealed that while both have equivalent nucleosome-stimulated ATPase activities, Isw1a and Isw1b differ in their abilities to bind to DNA and nucleosomal substrates, which possibly accounts for differences in specific activities in nucleosomal spacing and sliding. In vivo, the two Isw1 complexes have overlapping functions in transcriptional regulation of some genes yet distinct functions at others. In addition, these complexes show different contributions to cell growth at elevated temperatures (Vary, 2003).
Regulated binding of TBP to a promoter is a key event in transcriptional regulation. On glucose depletion, the S. cerevisiae Isw1 chromatin remodeling complex is required for the displacement of TBP from the PHO8 promoter, and thus mediating repression. Displacement of TBP also requires the sequence-specific bHLH-LZ factor Cbf1p that targets Isw1p to the PHO8 UAS. Cbf1p- and Isw1p-dependent displacement of TBP is also observed at the PHO84 promoter, but not at the ADH1 promoter, where loss of TBP is Cbf1p- and Isw1p independent. The results point to a promoter-specific Isw1p-dependent mechanism for targeted regulation of basal transcription by displacement of TBP from a promoter (Moreau, 2003).
Post-translational modification of nucleosomal histones has been suggested to contribute to epigenetic transcriptional memory. A case of transcriptional memory in yeast is described where the rate of transcriptional induction of GAL1 is regulated by the prior expression state. This epigenetic state is inherited by daughter cells, but does not require the histone acetyltransferase, Gcn5p, the histone ubiquitinylating enzyme, Rad6p, or the histone methylases, Dot1p, Set1p, or Set2p (see Drosophila Set2). In contrast, the ATP-dependent chromatin remodeling enzyme, SWI/SNF, is essential for transcriptional memory at GAL1. Genetic studies indicate that SWI/SNF controls transcriptional memory by antagonizing ISWI-like chromatin remodeling enzymes (Kundu, 2007).
Transcription by RNA polymerase I on nucleosomal templates requires binding of the transcription termination factor TTF-I to a cognate site 160 bp upstream of the transcription start site. Binding of TTF-I is accompanied by changes in the chromatin architecture which suggests that TTF-I recruits a remodeling activity to the rDNA promoter. A cDNA has been cloned that encodes TIP5 (TTF-I-interacting
protein 5), a 205 kDa protein that shares a number of important protein domains with WSTF (Williams syndrome transcription factor) and hAcf1/WCRF180, the largest subunits of human chromatin remodeling complexes hCHRAC and WCRF. TIP5 co-localizes with the basal RNA polymerase I transcription factor UBF in the nucleolus and is associated with SNF2h, the mammalian homolog of ISWI. The cellular TIP5-SNF2h complex, termed NoRC (nucleolar remodeling complex), induces nucleosome sliding in an ATP- and histone H4 tail-dependent fashion. The results suggest that NoRC is a novel nucleolar chromatin remodeling machine that may serve a role in the regulation of the rDNA locus (Strohner, 2001).
The marked conservation of ISWI homologs in species as diverse as yeast and humans suggests that they serve important functions. Apparently, the cell uses
different complexes to assemble specialized chromatin structures, and the different types of complexes may be targeted to different sets of cellular genes. The analysis of NoRC, a novel member of ISWI/SNF2h-based chromatin remodeling complexes, supports this view. The finding that NoRC both interacts with TTF-I and co-localizes with the basal Pol I transcription factor UBF suggests a function in rDNA transcription. Binding of TTF-I to its cognate
site upstream of the rDNA promoter induces chromatin remodeling, which is a prerequisite for Pol I transcription initiation. Thus, TTF-I
can activate transcription on chromatin templates, presumably by recruiting remodeling complexes to the rDNA promoter. Whether or not the TIP5 complex or
another remodeling machine that is present in the partially purified transcription system is responsible for TTF-I-mediated transcriptional activation is not yet known. The possibility that NoRC is associated with inactive ribosomal gene copies and exerts a repressive effect on Pol I transcription cannot be excluded. Further investigation of the role of NoRC on Pol I transcription using a defined in vitro system consisting of reconstituted nucleosomal arrays and purified transcription factors is clearly warranted. The reconstitution of NoRC from recombinant subunits will constitute a major milestone on this track. Regardless of whether NoRC exerts a stimulatory or repressive effect on rDNA transcription, NoRC has the potential to regulate cellular rRNA synthetic activity and, with that, ribosome biogenesis. Future studies will address the role of TIP5 in modulating transcriptional activity that occurs as the cell responds to external signals or progresses through the cell cycle (Strohner, 2001).
Chromatin remodeling can facilitate the recruitment of RNA polymerase II (Pol II) to targeted promoters, as well as enhancing the level of transcription. A further key role for chromatin remodeling in transcriptional termination is described. Using a genetic screen in S. pombe, the CHD-Mi2 class chromatin remodeling ATPase, Hrp1, has been identified as a termination factor. In S. cerevisiae, transcriptional termination and chromatin structure at the 3' ends of three genes all depend on the activity of the Hrp1 homolog, Chd1p, either alone or redundantly with the ISWI ATPases, Isw1p, and Isw2p. It is suggested that chromatin remodeling of termination regions is a necessary prelude to efficient Pol II termination (Alen, 2002).
Distinct forms of the yeast chromatin-remodeling enzyme Isw1p sequentially regulate each stage of the transcription cycle. The Isw1a complex (Iswlp/Ioc3p) represses gene expression at initiation through specific positioning of a promoter proximal dinucleosome, whereas the Isw1b complex (Iswlp/Ioc2p/Ioc4p) acts within coding regions to control the amount of RNA polymerase (RNAPII) released into productive elongation and to coordinate elongation with termination and pre-mRNA processing. These effects of Isw1b are controlled via phosphorylation of the heptad repeat carboxy-terminal domain (CTD) of RNAPII and methylation of the chromatin template. The transcription elongation factor Spt4p antagonizes Isw1p and overcomes the Isw1p dependent pausing of RNAPII at the onset of the elongation cycle. Overall these studies establish the central role played by Isw1p in the coordination of transcription (Morillon, 2003).
Chromatin plays an important role in transcriptional regulation and is generally considered to impede transcription initiation and elongation by RNA polymerase II (RNAPII). Chromatin structures over promoters are regulated by enzymes that covalently modify histones or alter chromatin by ATP-dependent disruption of DNA-histone interactions. These proteins are generally recruited to promoters by sequence-specific DNA binding proteins. During the elongation phase of transcription, RNAPII also uses a wide range of accessory factors to facilitate its movement through chromatin and several RNAPII-associated complexes have been identified. For example, in yeast, some complexes, such as PAF , Spt4/5, and TFIIS (Dst1p) are all associated with RNAPII throughout the elongation phase. However, other factors are localized to the 5' region of genes such as capping enzymes or to the 3' region of genes such as the cleavage/polyadenylation complex CF1A. Differential association between complexes and RNAPII appears to be a function of the heptapeptide repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) of the carboxy-terminal domain (CTD) of the largest subunit, Rpb1p, which is subject to differential phosphorylation. Hyperphosphorylation at Ser5 of the CTD by the Kin28p kinase subunit of TFIIH promotes disengagement of the enzyme from the promoter into the elongation phase of transcription coupled with the exchange of initiation factors for elongation factors. Ser5 phosphorylation then diminishes in the coding region while levels of Ser2 phosphorylation increase as RNAPII moves toward the 3' region. Significantly capping factors and the Set1p complex interact with phospho-Ser5 CTD while CF1A, and the Set2p histone methylase, interact with phospho-Ser2 CTD. Thus, the differential association of complexes to the CTD links the various phases of pre-mRNA synthesis, processing, and export. Furthermore, differential patterns of histone H3 methylation by Set1p (H3-K4) and Set2p (H3-K36) may respectively mark out the early and later stages of elongation and promote association of additional factors to the chromatin template to regulate these events. Candidates for this function are the FACT complex that facilitates transcription through chromatin, and the chromodomain containing chromatin-remodeling ATPase, Chd1p has been proposed to function as an elongation factor. Chd1p has also been identified as a termination factor at some yeast genes but at other genes Chd1p functions redundantly with the imitation switch (ISWI) chromatin-remodeling ATPases, Isw1p and Isw2p. Furthermore, a highly localized chromatin organization has been observed within the coding regions of a number of yeast genes that is dependent on the catalytic activity of Isw1p chromatin remodeling activity (Morillon, 2003 and references therein).
This study addresses the potential role played by Isw1p in the elongation and termination of transcription. Chromatin remodeling enzymes of the ISWI type are widely found in eukaryotes and are implicated in events leading to repression of expression. Thus, ISWI is associated with nontranscribed regions of polytene chromosomes in Drosophila and may displace TBP from promoters in Xenopus and yeast. However, a positive role for ISWI cannot be excluded as it is required for the expression of some genes in Drosophila. In addition, microarray data in yeast show that Isw1p and Isw2p, the two ISWI homologs, have both positive and negative effects on patterns of gene expression. Isw2p represses meiotic genes by configuring nucleosomes on promoters and may function in collaboration with the Sin3/Rpd3 histone deacetylase. Although less is known about the function of Isw1p, it is present in two distinct complexes, Isw1a and Isw1b, which confer distinct properties to the ATPase. Isw1a contains Ioc (isw one complex) 3p while Isw1b contains Ioc2p and Ioc4p. While Isw1a functions at promoters to repress genes, Isw1b possesses a bipartite function that coordinates both transcription elongation and termination (Morillon, 2003 and references therein).
Previous studies have implicated Isw1p in the repression of RNAPII transcribed genes. Isw1p is shown to display additional key functions in modulating the passage of RNAPII through all stages of gene transcription. This is achieved by separate Isw1p complexes: Isw1a (containing Ioc3p) and two functionally different forms of Isw1b containing either Ioc2p (for Iswi one complex 2, a PHD domain containing protein) or Ioc4p. Mutation of ISW1, encoding the common subunit of these three different complexes, has consequences at all stages of transcription. Instead, separate mutation of IOC3, IOC2, and IOC4 allows a functional dissection of the different roles that these Isw1p complexes play at each stage of the transcription cycle. The data demonstrate that the Isw1a complex (Isw1p/Ioc3p) is required for promoter inactivation while the Isw1b (Isw1p/Ioc4p/Ioc2p) correlates with the active state. Moreover, distinct positive (Ioc4p) and negative (Ioc2p) functions for the components of Isw1b are evident (Morillon, 2003).
Repression by the Isw1a complex is likely to be mediated by a local chromatin structure that prevents transcription factors, TBP or RNAPII, from associating with the promoter. A number of protein complexes containing Isw1p, Ioc3p, Mot1p, and Spt15 (TBP) have been identified and a role for Isw1p in mediating repression by aiding TBP displacement at the PHO8 promoter has recently been demonstrated (Morillon, 2003 and references therein).
The presence of Isw1p on inactive promoters ideally positions it to play a major role in regulating the switch to the activated state. During this switch, there may be an exchange of Ioc3p (in Isw1a) for Ioc2 and Ioc4p (in Isw1b) and a major change in the distribution of the Isw1p complex from the promoter to within the coding region of the gene. At this stage, there will also be an exchange of general initiation factors for the transcription elongation factors, marked by TFIIH (Kin28p)-dependent phosphorylation of Ser5 on the CTD of RNAPII. Support for the function of Isw1b early in transcription elongation comes from its requirement for the normal association of the Kin28p Ser5 CTD kinase with the promoter. In Drosophila, phosphorylation of Ser5 of the CTD is required for promoter proximal pausing of engaged RNAPII at heat shock promoters. In ioc2Δ and isw1K227R strains, where Ser5 CTD phosphorylation is defective, RNAPII is uncontrolled and accumulates at the 3' end of the gene. This links Ioc2p to Kin28 recruitment and the phosphorylation of Ser5 of the CTD and supports a role for Ioc2p in the accumulation of yeast RNAPII at promoter proximal positions. By contrast, Ioc4p controls Ser2 phosphorylation, H3K36 methylation, and recruitment of factors such as Rna15p for 3' end formation, events that coordinate cleavage and polyadenylation of the pre-mRNA. Moreover, Ioc2p and Spt4p are required to prevent premature Ser2 phosphorylation by Ioc4p. Thus, the data demonstrate that the Isw1b complex links Ser5 and Ser2 phosphorylation of the CTD on elongating RNAPII and coordinates the timing of these events. In addition, Ioc2p may promote release of RNAPII from the template. In ioc2Δ and isw1K227R mutants, disengaged RNAPII accumulates at the 3' end of the gene and there is the strong correlation between RNAPII with Ser5 CTD phosphorylation and the effective dissociation of RNAPII from the template. Thus, together Ioc2p and Ioc4p impose control on RNAPII allowing the coordination of transcription elongation and termination and effective cotranscriptional pre-mRNA processing including capping, 3' end cleavage, and polyadenylation. Even though capping is likely to be defective in isw1 mutants, since they fail to recruit Ceg1p, this is not expected to prevent transcript accumulation. Moreover, the exosome, associated with the degradation of improperly processed transcripts is linked to elongating RNAPII in Drosophila, and in yeast elongation may also be dependent on Isw1p function (Morillon, 2003).
Chromatin structure and chromatin remodeling activities are implicated in the control of transcription elongation. Localized recruitment of the SWI/SNF ATPase drives a remodeling reaction necessary for efficient transcript elongation in mammals. It is proposed that the Isw1p-positioned nucleosomes are central to control of transcript elongation in yeast. By this model, nucleosome +1 at MET16, positioned by Isw1p, impedes progress of RNAPII while the elongation factors, Spt4p and Dst1p, facilitate RNAPII movement through this nucleosome. Moreover, in isw1 mutants, RNAPII is no longer dependent on positive transcription elongation factors, such as Spt4p, and insensitive to drug induced arrest (Morillon, 2003).
It is striking that the local effect of Set1p trimethylation corresponds to two nucleosomes at the beginning of coding regions, very similar to the effect of Isw1p on promoter proximal chromatin structures observed in this study. Highly localized Isw1p-dependent chromatin structures and peaks of trimethylation are also associated within the 5' region of other yeast genes. Since the increase in H3 K4 trimethylation at 5' regions is dependent on Isw1p/Ioc4p, modifications to the histone proteins themselves may define this chromatin configuration. Moreover, Isw1p links events at the beginning and end of the transcription unit. It is well established that early elongation is marked by a combination of CTD Ser5 phosphorylation and H3K4 trimethylation by Set1p. These results now demonstrate that Isw1b coordinates these events with subsequent CTD Ser2 phosphorylation and H3K36 dimethylation by Set2p marking the termination phase of the transcription cycle. Thus, it is proposed that the Isw1p-dependent chromatin configuration acts as part of a promoter proximal transcription elongation checkpoint (TEC), involving components of the Isw1b complex and Spt4p, to coordinate the early and late events in the transcription cycle. The interplay of Ioc4p, Ioc2p, and Spt4p would control the amount of RNAPII prematurely aborted or released into productive elongation. Ioc4p and Spt4p may limit the propensity of Ioc2p to promote dissociation of RNAPII from the template at the checkpoint, allowing more RNAII into productive elongation. This would be entirely consistent with the positive roles proposed for Spt4p and Ioc4p in transcript elongation. In summary, it is proposed that Isw1p sequentially regulates each stage of the transcription cycle, linking events at the 5' and 3' end of the transcription unit and controlling the amount of RNAPII entering productive elongation (Morillon, 2003).
Members of the ISWI family of chromatin remodeling factors exhibit ATP-dependent nucleosome sliding, loading, and spacing activities in vitro. However, it is unclear which of these activities are utilized by ISWI complexes to remodel chromatin in vivo. This study sought to identify the mechanisms of chromatin remodeling by Saccharomyces cerevisiae Isw2 complex at its known sites of action in vivo. To address this question, a method was developed of identifying intermediates of the Isw2-dependent chromatin remodeling reaction as it proceeded. Isw2 complex is shown to catalyze nucleosome sliding at two different classes of target genes in vivo, in each case sliding nucleosomes closer to the promoter regions. In contrast to its biochemical activities in vitro, nucleosome sliding by Isw2 complex in vivo is unidirectional and localized to a few nucleosomes at each site, suggesting that Isw2 activity is constrained by cellular factors (Fazzio, 2003).
Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes was investigated. Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. This study shows that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. This analyses reveal how chromatin is organized on a global scale and advance understanding of how transcription is regulated (Whitehouse, 2007).
The positioning of thousands of nucleosomes adjacent to important regulatory sequences is controlled by Isw2. Yeast promoters frequently contain AT-rich DNA sequences that have been found to inhibit nucleosome positioning. Because Isw2 is able to use the energy from ATP hydrolysis to override the inherent nucleosome-positioning signal of the underlying DNA, Isw2 may function generally to reposition nucleosomes on unfavourable DNA sequences. Consistent with this, it was found that poly dA/dT tracts, which are highly enriched at nucleosome-free region (NFRs), are located within nucleosome +1 at many Isw2 targets. Loss of Isw2 would allow nucleosomes to adopt their inherent positioning preference, uncovering canonical or cryptic sites for transcriptional initiation. Because transcription is not necessary for the nucleosome positional changes caused by Isw2 deletion, transcription is likely to be a consequence rather than a cause of nucleosome repositioning at Isw2 targets. The broad scope of Isw2 action has implications for predictions of nucleosome positions on the basis of DNA sequence alone. These studies have had some success, but at present they are unable to predict accurately many nucleosome positions within the cell. The ability of proteins such as Isw2 to reposition nucleosomes provides a clear illustration that cellular factors actively operate to disrupt the intrinsic cues that would otherwise package the genome (Whitehouse, 2007).
The primary site of action of Isw2 at the 5' end of genes is the +1 nucleosome. This nucleosome is positioned such that the transcription start site is occluded by its 5' edge. Upstream of +1 generally lies a short NFR, which typically contains transcription-factor-binding sites and is probably the site for preinitiation-complex assembly. The nucleosome +1 generally contains the variant histone Htz1, which marks genes for rapid reactivation and is subject to rapid replication-independent turnover. This nucleosome is likely to act as a principal regulator of transcription, because RNA polymerase cannot reach the coding sequence without first transiting +1. In the context of this study, the specificity of Isw2 for this 'gatekeeper' nucleosome probably provides a regulatory mechanism to control gene expression by occluding the transcription start site or regulatory sequences through nucleosome repositioning (Whitehouse, 2007).
A key finding of this study is that transcription is able to initiate from cryptic start sites when ISW2 is deleted, which results in inappropriately oriented transcription from intergenic regions. This result is important because the mechanisms that ensure that transcription occurs in the correct orientation are largely unknown. These findings suggest a model in which promoters are not intrinsically directional and can support inappropriately oriented transcription when chromatin structure is perturbed. Thus, transcription factors and DNA sequence alone are insufficient to prevent initiation from cryptic sites at these promoters. Because Isw2 remodels chromatin structure at the 3' ends of many genes, the control of transcription by nucleosome positioning may be a general mechanism used by the cell (Whitehouse, 2007).
The class A, B and C synthetic multivulva (synMuv) genes act redundantly to
negatively regulate the expression of vulval cell fates in Caenorhabditis
elegans. The class B and C synMuv proteins include homologs of proteins
that modulate chromatin and influence transcription in other organisms similar
to members of the Myb-MuvB/dREAM, NuRD and Tip60/NuA4 complexes. To determine
how these chromatin-remodeling activities negatively regulate the vulval
cell-fate decision, a suppressor of the synMuv phenotype was isolated and it was found that the suppressor gene encodes the C. elegans homolog of
Drosophila melanogaster ISWI. The C. elegans ISW-1 protein
likely acts as part of a Nucleosome Remodeling Factor (NURF) complex with
NURF-1, a nematode ortholog of NURF301, to promote the synMuv phenotype.
isw-1 and nurf-1 mutations suppress both the synMuv
phenotype and the multivulva phenotype caused by overactivation of the Ras
pathway. These data suggest that a NURF-like complex promotes the expression of
vulval cell fates by antagonizing the transcriptional and chromatin-remodeling
activities of complexes similar to Myb-MuvB/dREAM, NuRD and Tip60/NuA4.
Because the phenotypes caused by a null mutation in the tumor-suppressor and
class B synMuv gene lin-35 Rb and a gain-of-function mutation in
let-60 Ras are suppressed by reduction of isw-1 function,
NURF complex proteins might be effective targets for cancer therapy (Andersen, 2006; full text of article).
Chromatin remodeling complexes have been implicated in the disruption or
reformation of nucleosomal arrays resulting in modulation of transcription, DNA
replication, and DNA repair. WCRF, a new chromatin-remodeling complex from HeLa cells, has been isolated. WCRF is composed of two subunits:
WCRF135, the human homolog of Drosophila ISWI, and WCRF180, a protein related to
the Williams syndrome transcription factor. WCRF180 is a member of a family of
proteins sharing a putative heterochromatin localization domain, a PHD finger,
and a bromodomain, prevalent in factors involved in regulation of chromatin
structure (Bochar, 2000).
Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI
were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the
only common component reported in these complexes. Purification of human
CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a
conserved subunit composition in completely different cell types, suggesting a
conserved function of ISWI. The human homologs of two novel
putative histone-fold proteins in Drosophila CHRAC (CHRAC-14 and CHRAC-16) are present in HuCHRAC. The
two human histone-fold proteins form a stable complex that binds naked DNA but
not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homolog of
Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as
a heterochromatin-targeting domain. hACF1 is a member of a family of proteins
with a related domain structure that all may target heterochromatin. A possible function for HuCHRAC in heterochromatin dynamics is discussed. HuCHRAC does not
contain topoisomerase II, which was reported earlier as a subunit of Drosophila
CHRAC (Poot, 2000).
A nucleosome remodeling and spacing factor, RSF, has been isolated and characterized. One of the RSF subunits is hSNF2h, a SNF2 homolog. Another hSNF2h-containing complex has been isolated and identified. A novel hSNF2h complex facilitates ATP-dependent chromatin assembly with the histone chaperone NAP-1. The complex possesses ATPase activity that is DNA-dependent and nucleosome-stimulated. This complex is capable of facilitating ATP-dependent nucleosome remodeling and transcription initiation from chromatin templates. In addition to hSNF2h, this complex also contains a 190-kDa protein encoded by the BAZ1A gene. Since both subunits are homologs of the Drosophila ACF complex (ATP-utilizing chromatin assembly and remodeling factor), this factor has been named human ACF or hACF (LeRoy, 2000).
The murine gene CHD1 (MmCHD1) was previously isolated in a search for proteins that would bind a DNA promoter element. The presence of chromo (chromatin organization modifier) domains (such as those found in Drosophila Brahma and Imitation SWI) and an SNF2-related helicase/ATPase domain (present also in Drosophila HP1 and Polycomb) led to speculation that this gene might regulate chromatin structure or gene transcription. Three novel human genes are
related to MmCHD1. Examination of sequence databases produce several more related genes, most of which are not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. A Drosophila homolog, DmCHD1, contains all the domains found in the human sequence; another homolog, DmCHD3, lacks the DNA-binding domain sequence. MmCHD1 preferentially binds via minor groove interactions to DNA that contains (A+T)-rich tracts including those in a matrix attachment region. The major region of sequence variation in CHD proteins is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains
being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggests that enhanced transcriptional arrest at RNA polymerase II pause sites (due to 6-azauracil-induced nucleotide pool depletion) is reduced in the deletion strain and that ScCHD1 inhibits transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related
helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template (Woodage, 1997).
A mammalian chromatin-associated protein, CHD1 (chromo-ATPase/helicase-DNA-binding domain), might have an important role in the modification of chromatin structure. The Drosophila melanogaster CHD1 homolog (dCHD1) encodes an 1883-aa open reading frame that is 50% identical and 68% similar to the mouse CHD1 sequence, including conservation of the three signature domains for which the protein was named. dCHD1 is related to both Drosophila Brahma and Imitation SWI as well as to Polycomb and HP1, based on the presence of both a helicase domain (found in Brahma and ISWI) and a chromo domain (found in Polycomb and HP1). When the chromo and ATPase/helicase domain sequences in various CHD1 homologs are compared with the corresponding sequences in other proteins, certain distinctive features of the CHD1 chromo and ATPase/helicase domains are
revealed. This suggests that CHD constitutes a distinct subgroup that diverged early in evolution from HP1 and PC subgroups as well as from ISWI type proteins. The dCHD1 gene maps to position 23C-24A on chromosome 2L. Western blot
analyses with antibodies raised against a dCHD1 fusion protein specifically recognize an approximately 210-kDa protein in nuclear extracts from Drosophila embryos and cultured cells. Most interestingly, these antibodies reveal that dCHD1 localizes to sites of extended chromatin (interbands) and regions associated with high transcriptional activity (puffs) on polytene chromosomes
from salivary glands of third instar larvae. These observations strongly support the idea that CHD1 functions to alter chromatin structure in a way that facilitates gene expression (Stokes, 1996).
A 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the yeast SWI/SNF complex. RSC, a second yeast chromatin remodeler, is at least 10-fold more abundant than SWI/SNF and is essential for mitotic growth. At least three RSC subunits are related to SWI/SNF peptides. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. No association of either RSC or SWI/SNF with RNA polymerase II holoenzyme was detected and no histone acetyltransferase activity was found. The functional distinction between SWI/SNF-related and NURF complexes corresponds with the classification of the ATPase components. The six chromatin-remodeling complexes so far described (yeast SWI/SNF, RSC, brahma complex, NURF, and the two mammalian SWI/SNF complexes) contain, respectively, the DNA-dependent ATPases Snf2/Swi2p, Sth1p, Brahma, ISWI, and mammalian Brg1p (or hBrm protein). All of these ATPases except ISWI are similar in their ATPase domains and in several additional regions, whereas their similarity to ISWI is limited to the ATPase domain alone. One such region, present in the carboxyl termini of all of the ATPases except ISWI, constitutes a bromodomain (Cairns, 1996).
Chromatin assembly and remodeling complexes alter histone-DNA interactions by using the energy of ATP hydrolysis catalyzed by nucleosome-dependent ATPase subunits. Several classes of ATP-dependent chromatin remodeling complexes exist, including the ISWI family. ISWI complexes disrupt histone-DNA interactions in vitro by facilitating nucleosome sliding. Snf2h is a widely expressed ISWI ATPase. The role of the Snf2h gene in mammalian development was investigated by generating a null mutation in mice. Snf2h heterozygous mutant mice are born at the expected frequency and appear normal. Snf2h-/- embryos die during the peri-implantation stage. Blastocyst outgrowth experiments indicate that loss of Snf2h results in growth arrest and cell death of both the trophectoderm and inner cell mass. To investigate the effect of decreased Snf2h levels in adult cells, antisense inhibition of Snf2h was performed in human hematopoietic progenitors. Reducing Snf2h levels inhibits CD34+ progenitors from undergoing cytokine-induced erythropoiesis in vitro. These results indicate that Snf2h is required for proliferation of early blastocyst-derived stem cells and adult human hematopoietic progenitors. Cells lacking Snf2h are thus prevented from further embryonic development and differentiation (Stopka, 2003).
A purified recombinant chromatin assembly system, including ACF (Acf-1 + ISWI) and NAP-1, has been used to examine the role
of histone acetylation in ATP-dependent chromatin remodeling. The binding of a transcriptional activator (Gal4-VP16) to chromatin
assembled using this recombinant assembly system dramatically enhances the acetylation of nucleosomal core histones by the
histone acetyltransferase p300. This effect requires both the presence of Gal4-binding sites in the template and the VP16-activation
domain. Order-of-addition experiments indicate that prior activator-meditated, ATP-dependent chromatin remodeling by ACF is
required for the acetylation of nucleosomal histones by p300. Thus, chromatin remodeling, which requires a transcriptional activator, ACF and ATP, is an early
step in the transcriptional process that regulates subsequent core histone acetylation. Glycerol gradient sedimentation and immunoprecipitation assays
demonstrate that the acetylation of histones by p300 facilitates the transfer of H2A-H2B from nucleosomes to NAP-1. The results from these biochemical
experiments suggest that (1) transcriptional activators (e.g., Gal4-VP16) and chromatin remodeling complexes (e.g., ACF) induce chromatin remodeling in the
absence of histone acetylation; (2) transcriptional activators recruit histone acetyltransferases (e.g., p300) to promoters after chromatin remodeling has occurred;
and (3) histone acetylation is important for a step subsequent to chromatin remodeling and results in the transfer of histone H2A-H2B dimers from nucleosomes
to a histone chaperone such as NAP-1. These results indicate a precise role for histone acetylation, namely to alter the structure of nucleosomes (e.g., facilitate the
loss of H2A-H2B dimers) that have been remodeled previously by the action of ATP-dependent chromatin remodeling complexes. Thus, transcription from
chromatin templates is ordered and sequential, with precise timing and roles for ATP-dependent chromatin remodeling, subsequent histone acetylation, and
alterations in nucleosome structure. The presence of altered (i.e., H2A-H2B-depleted) nucleosomes
at a transcriptionally active, chromatin-remodeled promoter may help to maintain an open chromatin structure conducive to multiple rounds of activated
transcription (Ito, 2000).
Using a 'crude' chromatin-based transcription system that mimics transactivation by RAR/RXR heterodimers in vivo, it was not possible to demonstrate that
chromatin remodeling is required to relieve nucleosomal repression. Using 'purified' chromatin templates, it has been shown that, irrespective of the presence
of histone H1, both ATP-driven chromatin remodeling activities and histone acetyltransferase (HAT) activities of coactivators recruited by liganded receptors
are required to achieve transactivation. DNA footprinting, ChIP analysis, and order of addition experiments indicate that coactivator HAT activities and two
ATP-driven remodeling activities are sequentially involved at distinct steps preceding initiation of transcription. Thus, both ATP-driven chromatin remodeling
and HAT activities act in a temporally ordered and interdependent manner to alleviate the repressive effects of nucleosomal histones on transcription by RARalpha/RXRalpha heterodimers (Dilworth, 2000).
The ATP requirement during the preincubation period preceding HeLa nuclear extract (NE) addition indicates the involvement of ATP-driven chromatin remodeling activity(ies) at an early stage in the process, leading to RAR/RXR-triggered transcriptional initiation. This involvement is strongly supported by the ATP requirement for heterodimers 'tight' binding to their chromatin cognate REs in the absence of any of the other components required to achieve efficient initiation of transcription. This tighter binding is indeed associated with a marked and selective disruption of the nucleosomal structure in the region encompassing the DR5 RE (direct repeat 5 response elements) to which RAR/RXR heterodimers are bound. It appears therefore that RAR/RXR heterodimers, although able to 'recognize' REs within a nucleosomal structure, cannot tightly bind them unless that structure is disrupted. Importantly, this 'tight' binding is ligand independent and does not require histone acetylation by p300/TIF2 coactivators (Dilworth, 2000).
Several ATP-driven remodeling complexes are present in 'crude' chromatin assembled in vitro using Drosophila extracts. NURF, CHRAC, and ACF complexes contain the dISWI ATPase subunit, while the Brahma ATPase subunit is present in dSWI/SNF. This latter complex was essentially removed through purification of 'crude' chromatin. Similar DNase I footprints were obtained in the presence of RARalpha/RXRalpha heterodimers with 'crude' (which contains ATP) and 'purified' (to which only ATP was added) chromatin preparations. The ability of purified hISWI-containing complexes, but not of purified hSWI/SNF complexes, to further enhance ATP-dependent footprints suggests that hISWI-containing complexes can mediate 'tight' binding of RAR/RXR heterodimers to their REs. This is consistent with studies showing a role for the dISWI-containing NURF complex in assisting binding of other transactivators to cognate REs. Thus, the 'weak' but clear footprint of heterodimers on their REs in the absence of ATP suggests that ISWI-catalyzed nucleosome remodeling activity(ies) results in a greater RE accessibility and therefore in 'tighter' binding. This initial 'weak' binding could be a limiting step, as maximal transcriptional initiation is achieved only upon heterodimer addition at the start of the preincubation period, whereas ATP can be added up to 20 min later with little decrease in transcription efficiency. Note in this respect that nucleosome remodeling by NURF is known to occur in vitro within minutes through short-range sliding, irrespective of histone H1 presence. Alternatively or concomitantly a limiting step may correspond to the possible recruitment/targeting of dISWI-containing complexes through interaction with RARalpha/RXRalpha heterodimers, as recently suggested in the case of progesterone receptor and demonstrated for SWI/SNF complexes recruited/targeted by a number of transactivators. It remains to be determined whether the hSNF2h(hISWI)-containing complexes, RSF and/or WCRF/hACF, could functionally substitute in this respect for the Drosophila ATP-driven remodeling activity that is responsible for 'tight' binding of RAR/RXR heterodimers. Human SWI/SNF chromatin remodeling complexes, which cannot be replaced by hSNF2h(hISWI)-containing complexes, are required at a later stage, as optimal transcription is still achieved upon hSWI/SNF addition at the same time as HeLa NE that provides the machinery required for Preinitiation complex (PIC) formation. In contrast, addition of hSWI/SNF just before NTPs is ineffective, indicating that some further chromatin remodeling is indeed required for PIC formation, but does not exclude additional effects on transcriptional elongation. Whether the effect of hSWI/SNF involves its targeting to the promoter region through direct or indirect recruitment by template-bound liganded heterodimers is unknown, but preliminary experiments have failed to reveal such interactions in immunoprecipitation assays using either purified components or F9 cell extracts. However, such a possibility is suggested by a number of in vivo and in vitro observations indicating that SWI/SNF complexes can be recruited through interaction with yeast or animal transactivators, including several NRs. Alternatively or concomitantly, hSWI/SNF may be preferentially recruited through the bromodomain of SNF2alpha/beta subunits by nucleosomes acetylated by coactivators, as bromodomains have been shown to exhibit a high affinity for acetylated lysine residues. This latter possibility may explain why in the absence of histone acetylation hSWI/SNF does not exert any stimulatory activity, unless it is added much before HeLa NE (Dilworth, 2000).
The second step in the process leading to transcriptional initiation triggered by RAR/RXR heterodimers corresponds to the ligand-dependent recruitment/targeting of coactivators that acetylates histones through intrinsic HAT activities. To be efficient, this step has to be preceded by the ATP-dependent ligand-independent receptor 'tight' binding step, and it appears to be a prerequisite for efficient PIC formation upon HeLa NE addition, since activation of transcription is strongly decreased when either Acetyl CoA, ligands, and/or p300/TIF2 is added at the same time as HeLa NE. This strongly suggests that the observed transcriptional activation can be attributed to histone acetylation but does not exclude that acetylation of either nonhistone chromatin proteins (components of the basal transcription machinery and/or activators) by coactivator acetyltransferases may play a role at a later stage(s) for enhanced PIC formation. In contrast, no stimulation of transcription by HeLa NE was observed on 'naked' cognate DNA templates in the presence of p300/TIF2, Acetyl CoA, and liganded heterodimers. Thus, nucleosomal histone acetylation by coactivators targeted to the promoter region through ligand-dependent recruitment by the receptors play a crucial role in transcriptional activation in vitro (Dilworth, 2000).
The human ISWI-containing factor RSF (remodeling and spacing factor) mediates nucleosome deposition and, in the presence of ATP, generates regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with
bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced
nucleosome arrays. Recombinant chromatin lacking histone acetylation is impaired in directing transcription. Histone-tail modifications regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 increases transcription. Methylation of the histone H3 tail by Suv39H1 represses transcription in an HP1-dependent manner. The effects of histone-tail
modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system is refractory to the effect imposed by acetylation and methylation (Loyola, 2001).
The reaction catalyzed by RSF appears mechanistically different from the reaction catalyzed by ACF, which requires the histone chaperone NAP-1. RSF appears to function stoichiometrically with DNA, whereas ACF functions catalytically. The assembly of regularly spaced nucleosomes, catalyzed by RSF, appears to proceed by at least two steps. The first step is nucleosome deposition, and it is likely mediated by the large subunit of RSF. This subunit (p325) is encoded by a novel gene. The amino acid sequence of p325 shows the presence of a large charged region, which might participate directly in the nucleosome assembly reaction. The small subunit of RSF, hSNF2h, does not interact directly with the histone octamer; however, RSF, like histone chaperones, interacts with the H3/H4 tetramer and the octamer. Moreover, the binding of RSF to DNA is dependent on the histone octamer. The interaction of RSF with the octamer is independent of the histone tails and does not require posttranslational modifications, since RSF interacts with the histone octamer generated with bacterially produced histones and with recombinant octamers deleted of the histone tails. Although, the nucleosome deposition step catalyzed by RSF is independent of the histone tails, the second step of the reaction, the ATP-dependent nucleosome spacing, is dependent on histone tails. Moreover, the efficiency of array formation is stimulated by p300-mediated acetylation. In agreement with previous studies showing that the Drosophila ISWI polypeptide requires the H4 tails for ATP-dependent nucleosome mobilization, the histone H4 tail is necessary for array formation. However, the histone H4 tail is not sufficient for the formation of regularly spaced nucleosome arrays, since the tails of the other histones influence array formation. p300-mediated stimulation of array formation requires acetylation of the tails of the H2A/H2B dimer. Surprisingly, however, the histone H3 tail negatively affects the p300-mediated stimulation of array formation. It is speculated that the negative effect imposed by the histone H3 tail might be overcome by other modifications, such as phosphorylation of histone H3 at Ser 10 or that an alternative HAT mediates acetylation of this tail. This remains to be elucidated (Loyola, 2001).
The establishment of conditions that permit the reconstitution of recombinant chromatin allows for the analysis of the effect of the different histone tail modifications in transcription. Toward this goal, the ability of the recombinant chromatin to be used as template for transcription was analyzed and the effect of
two histone-tail modifications was specifically analyzed: p300-mediated acetylation and Suv39H1-mediated methylation (Loyola, 2001).
Although these two modifications can have opposite effects on transcription, these modifications were not recognized in a highly reconstituted transcription system; their effect was observed only in crude extracts. There are different explanations for findings. The most logical explanation is that acetylation and/or methylation per se does not affect template utilization but affects the ability of the chromatin templates to be recognized by the transcription machinery. It is likely that these modifications provide marks on the
histone tails that are recognized by factors present in extracts but missing in the reconstituted system that affects transcription. This hypothesis is supported by the findings with methylation and transcription. It was found that HP1-mediated repression of transcription requires Suv39H1-mediated methylation of histone H3. This finding is in perfect agreement with results obtained in vivo showing that the binding of HP1 to chromatin requires methylation of histone H3-Lys 9. Surprisingly, however, chromatin, H3-Lys 9 methylation, and HP1 are
not sufficient to establish repression, since this could not be reproduced in a reconstituted transcription system. It is likely that other factors are required to establish repression. Studies in yeast have shown that histone deacetylation is required to establish the appropriate substrate for methylation by Suv39H1. Although the use of chromatin without pre-existing modification bypasses the requirement for the histone deacetylase enzymatic activity, it is possible
that the histone deacetylases that target histone H3-lysines 9 and 14 not only function to generate the appropriate substrate but also might be active components of the Suv39H1-repressive complex (Loyola, 2001).
With regards to acetylation, it was observed that chromatin reconstituted with hypoacetylated human histone polypeptides is not optimal for transcription in crude extracts; however, the reconstituted system is indifferent to acetylation of the histone polypeptides. This finding is in agreement with the histone-code hypothesis and strongly suggests that factors in the extract, but lacking in the reconstituted system, might recognize the
acetylated mark(s) to stimulate transcription. Using recombinant chromatin, it was observed that acetylation of histone tails, specifically by p300, stimulates transcription in extracts. In agreement with the results obtained using chromatin reconstituted with hypo/hyperacetylated human histones, no effect was observed in a reconstituted transcription system. Although a possible explanation to this observation is the absence of a factor in the reconstituted system, the inability of the reconstituted transcription system to respond to acetylation of the recombinant chromatin might also be the result of the inability of p300 to acetylate specific residues on the histone tails. The recombinant chromatin is devoid of histone-tail modifications, and it is likely that p300-mediated acetylation of a specific residue might require other
histone modifications. This possibility is supported by studies showing that phosphorylation of histone H3-Ser 10 modulates acetylation of histone H3-Lys
14. The presence of a specific kinase in the extract might phosphorylate histone H3-Ser 10, resulting in efficient acetylation. Elucidation of the factors necessary for p300-mediated acetylation to result in optimal transcription and of the factor(s) required for Suv39-H1-mediated methylation to result in repression of transcription, and their exact mechanism of action, require further studies. The development of the system described in the present study, capable of generating recombinant chromatin will permit the setting of biochemical complementation assays to isolate the different factors involved in these processes as well as the elucidation of their mechanism of action (Loyola, 2001).
Cloning by the transplantation of somatic nuclei into unfertilized eggs requires a dramatic remodeling of chromosomal architecture. Many proteins are specifically lost from nuclei, and others are taken up from the egg cytoplasm. Recreating this exchange in vitro, the chromatin-remodeling nucleosomal adenosine triphosphatase (ATPase) ISWI has been identified as a key molecule in this process. ISWI actively erases the TATA binding protein from association with the nuclear matrix. Defining the biochemistry of global nuclear remodeling may facilitate the efficiency of cloning and other dedifferentiation events that establish new stem cell lineages (Kikyo, 2000).
The Williams Syndrome Transcription Factor (WSTF), the product of the WBSCR9 gene, is invariably deleted in the haploinsufficiency Williams-Beuren Syndrome. Along with the nucleosome-dependent ATPase ISWI, WSTF forms a novel chromatin remodeling complex, WICH (WSTF-ISWI chromatin remodeling complex), which is conserved in vertebrates. The WICH complex was purified to homogeneity from Xenopus egg extract and was found to contain only WSTF and ISWI. In mouse cells, WSTF interacts with the SNF2H isoform of ISWI. WSTF accumulates in pericentric heterochromatin coincident with the replication of these structures, suggesting a role for WSTF in the replication of heterochromatin. Such a role is supported by the in vitro activity of both the mouse and frog WICH complexes: they are involved in the assembly of regular spaced nucleosomal arrays. In contrast to the related ISWI-interacting protein ACF1/WCRF180, WSTF binds stably to mitotic chromosomes. Since dysfunction of other chromatin remodeling factors often has severe effects on development, haploinsufficiency of WSTF may explain some of the phenotypes associated with this disease (Bozhenok, 2002).
Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catalysed by ISWI are converted into relocation of DNA relative to a histone octamer. Mammalian HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. Transient interaction of HMGB1 with nucleosomal linker DNA overlapping ISWI-binding sites enhances the ability of ACF to bind nucleosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity is inhibitory. These observations are consistent with a role for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling (Bonaldi, 2002).
The Imitation SWItch (ISWI) chromatin remodeling factors have been
implicated in nucleosome positioning. In vitro, they can mobilize nucleosomes bi-directionally, making it difficult to envision how they can establish precise translational positioning of nucleosomes in vivo. It has been proposed that they require other cellular factors to do so, but none has been identified thus far. This study demonstrates that both ISW2 and TUP1 are required to position nucleosomes across the entire coding sequence of the DNA damage-inducible gene RNR3. The chromatin structure downstream of the URS is indistinguishable in Deltaisw2 and Deltatup1 mutants, and the crosslinking of Tup1 and Isw2 to RNR3 is independent of each other, indicating that both complexes are required to maintain repressive chromatin structure. Furthermore, Tup1 represses RNR3 and blocks preinitiation complex formation in the Deltaisw2 mutant, even though nucleosome positioning is completely disrupted over the promoter and ORF. This study has revealed a novel collaboration between two nucleosome-positioning activities in vivo, and suggests that disruption of nucleosome positioning is insufficient to cause a high level of transcription (Zhang, 2004).
Two new subunits of the Isw2 chromatin-remodeling complex in Saccharomyces cerevisiae have been identified. Both proteins, Dpb4p and Yjl065cp (named Dls1p), contain histone fold motifs and are homologous to the two smallest subunits of ISWI-containing CHRAC complexes in higher eukaryotes. Dpb4p is also a subunit of the DNA polymerase epsilon (polepsilon) complex, and Dls1p is homologous to another polepsilon subunit, Dpb3p. Therefore, these small histone fold proteins may fulfill functions that are required for both polepsilon and Isw2 complexes. The role of Dls1p in known functions of the Isw2 complex was characterized in vivo. Transcriptional analyses reveal that the Isw2 complex requires Dls1p to various degrees at a wide variety of loci in vivo. Consistent with this, Dls1p is required for Isw2-dependent chromatin remodeling in vivo, although the requirement for this protein varies among Isw2 targets. Dls1p is likely required for functions of the Isw2 complex at steps subsequent to its interaction with chromatin, since a dls1 mutation does not affect cross-linking of Isw2 with chromatin (McConnell, 2004).
Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing. In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations. The epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase epsilon (Pol epsilon) or lacking one of the nonessential and histone fold motif-containing subunits of Pol epsilon, Dpb3 and Dpb4. A novel and putative chromatin-remodeling complex, ISW2/yCHRAC, has been identified that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4. Using the single-cell method developed in this study, it has been demonstrated that without Pol epsilon and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched. Furthermore, Pol epsilon and ISW2/yCHRAC function independently: Pol epsilon operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state. It is therefore proposed that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators (Iida, 2004).
Isw2 ATP-dependent chromatin-remodeling activity is targeted to early meiotic
and MATalpha-specific gene promoters in Saccharomyces cerevisiae. Unexpectedly,
preferential cross-linking of wild-type Isw2p was not detected at these loci.
Instead, the catalytically inactive Isw2p-K215R mutant is enriched at Isw2
targets, suggesting that Isw2p-K215R, but not wild-type Isw2p, is a sensitive
chromatin immunoprecipitation (ChIP) reagent for marking sites of Isw2 activity
in vivo. Genome-wide ChIP analyses confirmed this conclusion and identified tRNA
genes (tDNAs) as a new class of Isw2 targets. Loss of Isw2p disrupts the
periodic pattern of Ty1 integration upstream of tDNAs, but does not affect
transcription of tDNAs or the associated Ty1 retrotransposons. In addition to
identifying new Isw2 targets, these localization studies have important
implications for the mechanism of Isw2 association with chromatin in vivo.
Target-specific enrichment of Isw2p-K215R, not wild-type Isw2p, suggests that
Isw2 is recruited transiently to remodel chromatin structure at these sites. In
contrast, no evidence was found for Isw2 function at sites preferentially
enriched by wild-type Isw2p, leading to the proposal that wild-type Isw2p
cross-linking reveals a scanning mode of the complex as it surveys the genome
for its targets (Gelbart, 2005).
The modification of chromatin structure is an important regulatory mechanism for developmental gene expression. Differential expression of the mammalian ISWI genes, SNF2H and SNF2L, has suggested that they possess distinct developmental roles. This study describes the purification and characterization of the first human SNF2L-containing complex. The subunit composition suggests that it represents the human ortholog of the Drosophila nucleosome-remodeling factor (NURF) complex. Human NURF (hNURF) is enriched in brain, and it regulates human Engrailed, a homeodomain protein that regulates neuronal development in the mid-hindbrain. Furthermore, hNURF potentiates neurite outgrowth in cell culture. Taken together, these data suggess a role for an ISWI complex in neuronal growth (Barak, 2003).
The Drosophila ISWI protein exists in three multiprotein complexes, namely, ACF, CHRAC and NURF. Mammalian complexes corresponding to ACF and CHRAC have been purified and contain the SNF2H protein. Additional unique mammalian ISWI complexes have also been purified, including RSF, WICH, NoRC and SNF2H-cohesin, and these all comprise the SNF2H protein. Despite the growing list of mammalian ISWI complexes, a NURF equivalent or complexes containing the related protein SNF2L has been notably absent. The hNURF complex identified in this study contains BPTF and RbAP46/48. Surprisingly, hNURF does not contain the inorganic pyrophosphatase protein NURF38. Nonetheless, the biochemical activity of hNURF is similar; it displays predominantly nucleosome-stimulated ATPase activity, as well as potent chromatin-remodeling activity on oligonucleosomal arrays (Barak, 2003).
The brain-enriched expression profile of SNF2L prompted an examination of a role for hNURF in neuronal physiology. SNF2L chromatin-remodeling activity can induce neurite outgrowth in a tissue culture-based assay, and this is specific to SNF2L-containing ISWI complexes since SNF2H expression does not result in a similar induction. The conversion of a neuroblast to a differentiated neuron will require the modification of chromatin structure at numerous genes, for both activation and repression, and it is not likely to be restricted to the NURF complex. Nonetheless, these studies suggest that hNURF has a role in this process and, thus, identification of target genes will help elucidate the molecular pathways. In this regard, hNURF can regulate the mammalian engrailed genes, through a direct interaction at the promoters of these two homeotic loci. The murine engrailed genes are critical regulators of mid-hindbrain development; ablation leads to animals that are missing most of the colliculi and cerebellum. Although engrailed was identified previously as a NURF target gene through the characterization of flies harboring mutant ISWI or NURF301 genes, a neural defect was not appreciated due to the early lethality of these animals. As such, this may represent a novel function for the NURF complex (Barak, 2003).
The effect of chromatin-remodeling complexes on development is a well-established phenomenon. Linkages between chromatin remodeling and developmental disorders include ATRX and mental retardation, SMARCAL1 and Schimke immuno-osseous dysplasia, CSB and Cockayne syndrome, and SNF2H and Williams syndrome. It is hypothesized that the hNURF complex may represent another connection of a chromatin-remodeling protein to disorders of development, and it can be stated with confidence that the hNURF complex regulates other developmentally important genes. In this regard, the analysis of flies ablated for the NURF complex also suggests a role for the hNURF complex in hematopoietic development and the regulation of chromosome structure. However, such studies in mammals must await further dissection using in vivo model systems (Barak, 2003).
Chromatin dynamics play an essential role in regulating DNA transaction processes, but it is unclear whether transcription-associated chromatin modifications control the mRNA ribonucleoparticles (mRNPs) pipeline from synthesis to nuclear exit. This study identified the yeast ISW1 chromatin remodeling complex (see Drosophila ISWI) as an unanticipated mRNP nuclear export surveillance factor that retains export-incompetent transcripts near their transcription site. This tethering activity of ISW1 requires chromatin binding and is independent of nucleosome sliding activity or changes in RNA polymerase II processivity. Combination of in vivo UV-crosslinking and genome-wide RNA immunoprecipitation assays show that Isw1 and its cofactors interact directly with premature mRNPs. These results highlight that the concerted action of Isw1 and the nuclear exosome ensures accurate surveillance mechanism that proofreads the efficiency of mRNA biogenesis (Babour, 2016).
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