Histone H1
H1 histones, found in all multicellular eukaryotes, are associated with linker DNA between adjacent nucleosomes. Presumably this is to keep the
chromatin in a compact, helical state. The identification of multiple histone H1 subtypes in vertebrates suggests these proteins have
specialized roles in chromatin organization and thus influence the regulation of gene expression in the multicellular organism. The
mechanism by which the association of H1 with nucleosomal DNA is regulated is not completely understood, but affinity for different
DNA sequences may play a role. A specific H1 subtype in the mouse, namely H1b, selectively binds to a regulatory
element within the protein-encoding sequence of a replication-dependent mouse Histone 3.2 gene. This coding
region element, Omega, is the target of very specific interactions in vitro with another nuclear factor, termed the Omega factor. This
element is required for normal gene expression in stably transfected rodent cells. The mouse Histone 1b protein interacts poorly (100-fold
lower affinity) with the comparable Omega sequence of a replication-independent mouse Histone 3.3 gene. This H3.3 sequence differs in
only 4 out of 22 nucleotide positions from the H3.2 sequence. These findings raise the possibility that this H1b protein plays a specific
role in regulation of expression of the replication-dependent histone gene family (Kaludov, 1997).
An experimental assay was developed to search for proteins capable of antagonizing histone H1-mediated general repression of transcription. T7 RNA polymerase templates containing an upstream scaffold-associated region were highly selectively repressed by H1 relative to non-SAR control templates. This is due to the nucleation of H1 assembly into flanking DNA brought about by the numerous A-tracts (AT-rich sequences containing short homopolymeric runs of dA.dT base pairs) of the SAR. A partial, selective titration of these A-tracts by the high mobility group (HMG) protein HMG-I/Y leads to the complete derepression of transcription from the SAR template by inducing the redistribution of H1 onto non-SAR templates. SARs are associated with many highly transcribed and regulated genes, where they may serve to facilitate the HMG-I/Y-mediated displacement of histone H1 in chromatin. Indeed, HMG-I/Y was found to be strongly enriched in the H1-depleted subfraction that can be isolated from chromatin (Zhao, 1993).
In replicating
molecules the passage of the replication machinery destabilizes the nucleosomal organization of the
chromatin fiber over a distance of 650 to 1100 bp. In front of the replication fork an average of two nucleosomes are
destabilized, presumably by the dissociation of histone H1 and the advancing replication machinery. On
daughter strands, the first nucleosome is detected at a distance of about 260 nucleotides from the elongation
point. This nucleosome is interpreted to contain no histone H1, while no stepwise association of (H3-H4)2
tetramers with H2A/H2B dimers on nascent DNA can be detected in vivo. The second nucleosome after the
replication fork appears to contain histone H1. The prolonged nuclease sensitivity of newly replicated
chromatin described in the literature therefore may not be due to a slow reassociation of histone H1 (Gasser, 1996).
Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin
structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a
transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a
model system, it has been demonstrated that the nonhistone chromosomal protein HMG-14, which is
known to preferentially associate with active chromatin, completely alleviates histone H1-mediated
inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent
compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of
HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural
activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions
from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is
involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of
mammalian genes (Ding, 1997).
Upstream binding factor (UBF) is a vertebrate RNA polymerase I transcription factor that can bend
and wrap DNA. To investigate the likely role of UBF as an architectural protein for rRNA genes that are organized
in chromatin, an examination was carried out of UBF's ability to bind rRNA gene enhancers assembled into nucleosome cores
(DNA plus core histones) and nucleosomes (DNA plus core histones plus histone H1). UBF binds
with low affinity to nucleosome cores formed with enhancer DNA probes of 162 bp. However, on
nucleosome cores which contain approximately 60 bp of additional linker DNA, UBF binds with
high affinity, similar to its binding to naked DNA, forming a ternary DNA-core histone-UBF complex.
UBF could be stripped from ternary complexes with competitor DNA to liberate nucleosome cores,
rather than free DNA, suggesting that UBF binding to nucleosome cores does not displace the core
histones H2A, H2B, H3, and H4. DNase I, micrococcal nuclease, and exonuclease III footprinting
suggests that UBF and histone H1 interact with DNA on both sides, flanking the histone octamer.
Footprinting shows that UBF outcompetes histone H1 for binding to a nucleosome core and will
displace, if not dissociate, H1 from its binding site on a preassembled nucleosome. These data suggest
that UBF may act to either prevent or reverse the assembly of transcriptionally inactive chromatin structures
catalyzed by linker histone binding (Kermekchiev, 1997).
BALB/c 3T3 cell lines containing integrated copies of the MMTV promoter driving
a reporter gene were constructed. Expression vectors in which either of two H1
variants, H10 or H1c, were under control of an inducible promoter were
introduced into these lines. Surprisingly, overproduction of either variant
results in a dramatic increase in basal and hormone-induced expression from the
MMTV promoter. H1 overproduction also slows the loss of MMTV promoter activity
associated with prolonged hormone treatment. Transiently transfected MMTV
reporter genes, which do not adopt a phased nucleosomal arrangement, do not
display increased activity upon H1 overproduction. Thus the effects observed for
stable constructs most likely represent a direct effect of H1 on a
chromatin-mediated process specific to the nucleosomal structure of the
integrated constructs. Induction of increased levels of acetylated core histones
by treatment with trichostatin A also potentiates MMTV activity and this effect
is additive to that caused by H1 overproduction. However, the effects of TSA
treatment, in control or H1-overproducing cells, were eliminated by inhibiting
protein synthesis. TSA treatment does not necessarily potentiate MMTV promoter
activity by increasing core histone acetylation within the MMTV promoter but
perhaps by altering the synthesis of an unlinked transcriptional regulator (Gunjan, 1999a).
Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns (Fan, 2005: full text of article).
In contrast to the studies in yeast, in which no common features of the H1-sensitive genes were found, the current results identify a category of genes whose expression is especially sensitive to H1 content. Nearly one-third of the genes with altered expression in the H1-depleted cells are thought to be normally regulated by DNA methylation. Importantly, 4 of the 29 known genes (Xlr3, Pem, H19, and Igf2) identified in this study have an altered expression in mouse embryonic fibroblasts deficient for Dnmt1, the major maintenance DNA methyltransferase in mammalian cells. The level of DNA methylation was measured within the H19-Igf2 imprinting control region (ICR) that regulates the reciprocal expression of the two genes at this locus. The analysis showed that many of the CpG dinucleotides in this region were undermethylated in H1-depleted ES cells compared with control ES cells. This observation, along with the finding that expression of H19 is upregulated and that of Igf2 is downregulated in the H1-depleted cells, is entirely consistent with the current model for control of the two genes by the ICR. Reduced CpG methylation was also found within several DMRs at the Gtl2-Dlk1 imprinted locus, and the expression of Gtl2, like H19, is significantly upregulated in H1-depleted cells. The effect of H1 depletion on DNA methylation within the imprinting control regions of these two loci is specific since it was found that the level of CpG methylation in bulk DNA, within the major and minor satellite DNA sequences and within endogenous C type retrovirus repeats, is not altered in the triple-H1 null ES cells. CpG methylation levels also were not altered in the promoter regions of the α-actin and the Myl-c genes, whose expression was not affected by H1 depletion. It is suggested that the effect of H1 on DNA methylation in the two imprinting control regions is direct because H1 is indeed reduced by about one-half in these regions in the triple-H1 null ES cells. These results point to a previously unrecognized contribution of H1 to the control of gene expression in mammalian cells, namely through an effect on DNA methylation. In contrast, elimination of an H1-like gene in the filamentous fungus Ascobolus immerses was reported to have no effect on methylation-associated gene silencing, although it did lead to global hypermethylation, an effect that was not observe in mammalian cells. Recently, downregulation of H1 levels in Arabidopsis thaliana was reported to lead to both minor increases and decreases in the methylation patterns of certain repetitive and single-copy DNA sequences. Therefore, H1 may play a role in regulating specific patterns of DNA methylation in both plants and animals (Fan, 2005).
Although changes in DNA methylation patterns at specific loci likely account for a substantial number of the gene-expression changes observed in H1-depleted cells, it seems likely that other mechanisms are also involved. For example, the finding that 2 of the 19 genes that were upregulated encode H1 linker histones themselves suggests the existence of a feedback mechanism within the H1 gene family. The development of a cell-culture system in which highly specific changes in gene expression occur in response to H1 levels should allow a deeper understanding of the mechanisms by which linker histones participate in control of gene expression and possibly other aspects of chromatin regulation. The reintroduction of H1 genes into these cells also should make possible studies of linker-histone structure-function relationships in gene regulation (Fan, 2005).
Histone H1 preferentially and cooperatively binds scaffold-associated regions (SARs) in vitro via specific
interactions with the numerous short A + T-rich tracts (A-tracts) contained in these sequences. Selective
titration of A-tracts by the oligopeptide distamycin abolishes this interaction and results in a redistribution of
H1. Similarly, treatment of intact cells and isolated nuclei with distamycin specifically enhances cleavage of
internucleosomal linkers of SARs by topoisomerase II and restriction enzymes. The increased accessibility of
these linkers is thought to result from the unfolding (or opening) of the chromatin fiber and to be due to a
reduced occupancy by histone H1. Chromatin extraction and H1 assembly experiments support this view. It is thought that H1-depleted chromatin regions may be generated by titration of A-tracts by
putative distamycin analogues; this local opening may spread to adjacent regions assuming highly cooperative
H1-H1 interactions in chromatin (Kas, 1993).
Tandemly repeated DNA sequences of (GGA:TCC)n are found in tracts up to 50 base pairs long,
dispersed at thousands of sites throughout the genomes of eukaryotes. The formation of complexes paired between two DNAs containing such repeats occurs in vitro; enhancement of the pairing is facilitated by glutathione S-transferase fusion proteins of high mobility group protein 1
and histone H1. This assembly depends on incubation time at 37 degrees C and concentrations of the
proteins and DNA; the enhancement is inhibited by distamycin and actinomycin D interacting with
DNA through the minor groove. The structure of the DNA-DNA complex is deduced by comparison of its
mobility in gel electrophoresis with those of synthetic markers of heterotetramers. Three synthetic and
genomic DNA fragments containing repeats that have different arrangements exhibit different
efficiencies of DNA pairing, implying that the pairing is affected by the number of repeat units and the
arrangement of repeats in a sequence. Intriguingly, pairing occurs between homologous fragments but
not between heterologous DNAs among the three. These results suggest that the repeat-mediated
DNA pairing plays a role in the organization of higher order architecture of chromatin and possibly
chromosome segregation, requiring sequence-specific association events of DNA molecules (Mishima, 1997).
The chromatin structure induced by the phosphorylation of histone
H1 influences the replication efficiency of SV40 minichromosomes in vitro. Salt-treated SV40 minichromosomes were reconstituted with differentially phosphorylated forms of histone
H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies reveal a clear difference between
minichromosomes reconstituted with S-phase histone H1 as compared with histone H1 from G0- or M-phase cells,
indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using
reconstituted minichromosomes as substrate in the SV40 in vitro replication system, a higher
replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 was found, as compared with G0- or
M-phase histone H1 (Halmer, 1996).
The nucleosomal organization, histone H1 subtypes, and histone H1 phosphorylated isoforms have been compared in ras-transformed and parental 10T1/2 mouse fibroblasts. Ras-transformed mouse fibroblasts have a less condensed chromatin structure than normal fibroblasts.
Ras-transformed and parental 10T1/2 cells have similar amounts of H1 subtypes, proteins that play a key role in the
compaction of chromatin. However, interphase ras-transformed cells have higher levels of
phosphorylated H1 isoforms than parental cells. G1/S phase-arrested ras-transformed cells have higher amounts of
phosphorylated H1 than G1/S phase-arrested parental cells. Mouse fibroblasts transformed with fes, mos, raf,
myc, or constitutively active mitogen-activated protein (MAP) kinase kinase manifest increased levels of
phosphorylated H1. These observations suggest that increased phosphorylation of H1 is one of the
consequences of the persistent activation of the mitogen-activated protein kinase signal transduction pathway.
Phosphorylated H1b is localized in centers of RNA splicing in the
nucleus, suggesting that this modified H1 subtype is complexed to transcriptionally active chromatin (Chadee, 1995).
The importance of histone H1 heterogeneity and total H1 stoichiometry in
chromatin has been enigmatic. A detailed characterization of the
chromatin structure of cells overexpressing either H1(0) or H1c is reported. Nucleosome spacing was found to change during cell cycle progression, and overexpression of
either variant in exponentially growing cells results in a 15-base pair increase
in nucleosome repeat length. H1 histones can also assemble on chromatin and
influence nucleosome spacing in the absence of DNA replication. Overexpression
of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion
of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1
variants, it has been shown that micrococcal nuclease-resistant chromatin is specifically
enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance
of a unique mononucleosome species of higher mobility on nucleoprotein gels.
Domain switch mutagenesis reveals that either the N-terminal tail or the
central globular domain of the H1(0) protein can independently give rise to
this unique mononucleosome species. These results in part explain the
differential effects of H1(0) and H1c in regulating chromatin structure and
function (Gunjan, 1999b).
Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. In P19 embryonal carcinoma (EC) nuclei, HP1alpha, beta and gamma form homo- and
heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless
nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1 interacts with the linker histone H1. HP1 binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1 and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly (Nielsen, 2001).
Despite the key role of the linker histone H1 in chromatin
structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. This work has used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution hydroxyl radical footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. It was also found that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 is required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA (Syed, 2010).
NF-kappaB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. This study used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A-H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, it was found that NF-κB is able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-kappaB dependent genes (Lone, 2013).
Most studies of gene induction by inflammatory stimuli have focused on transcription factors that recognize specific DNA sequences and the cytoplasmic events that regulate the activation of these transcription factors. However, transcriptional activation of eukaryotic genes is also influenced by chromatin structure. Studies on the alterations in the chromatin structure required for productive NF-κB binding are essential for understanding the control of expression of inflammatory genes. However, the available data on this important topic are scarce and contradictory. This study used a combination of EMSA, hydroxyl radical (.OH) and UV laser footprinting to analyze how NF-κB binds to nucleosomes and the effect of histone H1 on the binding. The data provide definitive evidence that NF-κB is able to bind specifically to its cognate sequence when inserted at the end of the nucleosome, but not when it was inserted in vicinity to the nucleosome dyad. The accessibility to the ends of the nucleosome could be explained by the weaker histone-DNA interactions at these sites and their spontaneous unwrapping. At the center (the dyad) of the nucleosome, where the histone-DNA interactions are very strong, NF-κB is unable to specifically bind its cognate site. By contrast, several studies in the past have reported that some transcription factors, including NF-κB, were able to invade the nucleosome and to bind to nucleosome-embedded recognition sequences even when located in the center of nucleosomal DNA. However, these studies were carried out at low nucleosome concentrations at which sub-nucleosomal (hexameric and tetrameric) particles tend to appear. In order to understand whether the nucleosomes per se act as barriers for transcription factor binding, it is imperative to have homogenous population of nucleosomes devoid of any sub-nucleosomal entities. These sub-nucleosomal entities are formed by the loss of one or both H2A-H2B dimers and hence contain disorganized nucleosomal DNA which most likely would permit the specific binding of transcription factors. The results demonstrate that eviction of H2A/2B dimers is required for the binding of NF-κB. This can be achieved by the binding of factors that can disrupt the nucleosomes either directly by themselves indirectly through the recruitments of other nucleosome disrupting activity (Lone, 2013).
It has also been reported that the remodeling of 156 bp nucleosome core particles by SWI-SNF leads to complete and specific binding of NF-κB to its binding sites buried inside the nucleosome. However, this study observed only partial binding of NF-κB at the nucleosomal dyad when the nucleosomes are repositioned by the ATP dependent remodeler RSC. This discrepancy could be attributed to partial histone eviction under the very high concentration of the SWI-SNF that was used to remodel the nucleosomes and/or a presumable instability of the non-canonical (loss of the dyad axis) slid core particles (Lone, 2013).
Unexpectedly, in contrast to the partial accessibility to dimeric restriction enzymes at the dyad and efficient base excision repair (BER) initiatio remosomes did not show specific binding of NF-κB. Thus, in line with the available structural information , the specific binding of NF-κB requires much higher perturbations in histone-DNA interactions and unpeeling of its cognate sequence from the histone surface allowing it to 'embrace' DNA and to productively bind to it. The experimental results further demonstrate that such specific and productive binding could be efficiently achieved when the H2A–H2B dimer is removed from the nucleosome or when the histone octamer is repositioned in a way that the binding site nears the edge (Lone, 2013).
The compaction of chromatin by the linker histone in general has a global and repressive impact on transcription. Linker histone H1 brings the two helices close to each other and leads to the formation of a so-called 'stem' structure (Syed, 2010). Binding of H1 to DNA at the termini of nucleosomes inhibit spontaneous wrapping and unwrapping of DNA and hence would prevent the binding of transcription factors. Another possibility in which H1 could affect transcription is by occupying the binding sequences of those transcription factors whose binding sites are located in the linker region. This suggests that TF will have to compete with H1 to bind to their cognate sites. Several studies have provided the evidence that in certain cases linker histone can be directly displaced by transcription factor. In agreement with these studies, this study found that the presence of histone H1 does not prevent the specific binding of NF-κB when their binding regions overlap. In fact, NF-κB binding completely displaces histone H1 from the nucleosomes. It was also observed that H1 cannot displace the specifically bound NF-κB. In vivo, this competition might be even more in favor of NF-κB as H1 is quite mobile and dynamic (Lone, 2013).
The in vitro data sheds light on the in vivo requirements for the alterations in chromatin structure necessary for the productive binding of NF-κB. These include either a removal of H2A-H2B dimers from the nucleosome and/or chromatin remodeler induced mobilization of the histone octamer. The H2A-H2B dimers are more easily displaced from the histone core than H3 and H4 and they are extensively exchanged in vivo. Moreover, in mammalian cells the nucleosomes in the vicinity of the TSS contain the histone variant H2A.Z. A tentative hypothesis is that specific chaperones, recognizing variant H2A.Z nucleosomes, could be involved in the removal of H2A.Z-H2B variant dimer, thus allowing binding of the NF-κB transcription factors to any site of the nucleosomal DNA (Lone, 2013).
Both cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) and trans-diamminedichloroplatinum(II)
form covalent adducts with DNA. However, only the cis isomer is a potent anticancer agent. It has been
postulated that (1) the selective action of cis-DDP occurs through specific binding of nuclear proteins to
cis-DDP-damaged DNA sites and that (2) binding blocks DNA repair. A very abundant nuclear
protein, the linker histone H1, binds much more strongly to cis-platinated DNA than to trans-platinated or
unmodified DNA. In competition experiments, H1 is shown to bind much more strongly than HMG1,
which had been previously considered a major candidate for such binding in vivo (Yaneva, 1997).
PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. This study describes previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed chromatin structures. PARP-1 binds in a specific manner to nucleosomes and modulates chromatin structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of chromatin and a modulator of chromatin structure through its intrinsic enzymatic activity (Kim, 2004).
The similarities between the effects of PARP-1 and H1 on chromatin prompted a determination if PARP-1 and H1 might bind to overlapping sites on nucleosomes. In glycerol gradient sedimentation analyses, H1 promotes the formation of compact, faster migrating chromatin structures like PARP-1. Competitive binding between PARP-1 and H1 was examined using a 4-fold excess of H1 so that competition could be readily observed. When added to preassembled PARP-1-containing chromatin, H1 completely displaced the PARP-1 and was itself incorporated into the chromatin. Similar results were observed when a 4-fold excess of PARP-1 was used to compete with H1. Together, these results indicate that PARP-1 and H1 bind competitively to overlapping sites on nucleosomes (Kim, 2004).
To explore the relationship between PARP-1 and H1 in cells, a mononucleosome immunoprecipitation assay was used. In this assay, formaldehyde-crosslinked chromatin from HeLa cells was digested extensively with MNase to yield mononucleosomes and then subjected to immunoprecipitation with antibodies to PARP-1 or H1. The presence of PARP-1, H1, and core histones in the immunoprecipitated material was determined by Western blotting. Under conditions where approximately equal amounts of core histones, which serve as an internal control in this assay, were coprecipitated by the PARP-1 and H1 antibodies, PARP-1 was depleted from the H1 immunoprecipitates and H1 was depleted from the PARP-1 immunoprecipitates. These results suggest that PARP-1 and H1 reside in distinct nucleosomal fractions in cells (Kim, 2004).
Analysis of the coprecipitated DNA fragments revealed average sizes of ~160 bp for the PARP-1 coprecipitated DNA and ~165 bp for the H1 coprecipitated DNA, compared to an average size of ~150 bp for DNA from the bulk input mononucleosomes. In addition, more subnucleosomal DNA fragments were observed with the bulk input mononucleosomes, indicating that the DNA in that population of mononucleosomes was generally more accessible than the DNA in the PARP-1- and H1-enriched mononucleosomes. These results using native chromatin are nearly identical to the results obtained for the in vitro 'chromatosome stop' assay. Thus, similar effects of PARP-1 and H1 on nucleosomal repeat length and nuclease sensitivity are observed in vivo and in vitro (Kim, 2004).
The relationship between PARP-1 and H1 further examined by immunofluorescent staining of Drosophila salivary gland polytene chromosomes for PARP-1 and H1. This approach provides a global view of PARP-1 and H1 distribution over an entire genome from interphase cells. Both PARP-1 and H1 showed a broad distribution on the polytene chromosomes, but the pattern of PARP-1 staining was distinct from the pattern of H1 staining. The distinct pattern of staining for PARP-1 and H1 is most evident in the merged image, where areas of overlap, if present, would show up as yellow. When the distribution of PARP-1 and H1 was compared with highly condensed chromatin bands, PARP-1 was present in the less compact chromatin interbands, while H1 showed a nearly complete overlap with the highly condensed chromatin bands. These results indicate that PARP-1 and H1 reside in distinct chromatin domains and contribute to the formation of distinct higher-order chromatin structures in cells (Kim, 2004).
PARP-1, the prototypical and most abundantly expressed member of a family of PARP proteins, has been implicated in the regulation of chromatin structure and transcription (Kraus, 2003). PARP-1 possesses an intrinsic enzymatic activity that catalyzes the transfer of ADP-ribose units from donor NAD+ molecules to target proteins as monomers, oligomers, or polymers of ADP-ribose. However, compared to other chromatin regulatory proteins with enzymatic activities (e.g., histone-modifying enzymes, chromatin remodeling complexes), PARP-1 is poorly understood. PARP-1 has a highly conserved structural and functional organization, including (1) an N-terminal DNA binding domain containing two zinc-finger motifs, (2) a central automodification domain containing a BRCT ('BRCA1 C terminus-like') motif, and (3) a C-terminal NAD+ binding catalytic domain. PARP-1's enzymatic activity is stimulated dramatically by the binding of PARP-1 to damaged DNA (e.g., double-strand breaks) and hence, most studies of PARP-1 to date have focused on its role in DNA repair and cell death pathways. Considerably less is known about the chromatin-dependent gene regulatory activities of PARP-1 under physiological conditions where the integrity of the genome is maintained (Kim, 2004 and references therein).
A number of studies have examined PARP-1 as a regulator of chromatin structure and transcription, but conflicting results have left the role of PARP-1 unclear. Two modes of PARP-1 regulatory activity have been proposed: (1) a histone-modifying enzymatic activity that can regulate chromatin structure and (2) an enhancer/promoter binding cofactor activity that can act in conjunction with other transcription-related factors (Kraus, 2003). With regard to the regulation of chromatin, early studies have shown that purified PARP-1 could (ADP-ribosyl)ate chromatin proteins (e.g., H1 and, to a lesser extent, core histones), promoting the decondensation of chromatin and destabilization of nucleosomes. Further studies suggested a role for polyanionic poly(ADP-ribose) (PAR) itself, either attached to proteins or as a free polymer, as a core histone binding matrix that can act as a histone acceptor to further destabilize nucleosomes. More recent studies have demonstrated PARP-1-dependent accumulation of PAR at decondensed, transcriptionally active loci in native chromatin (Tulin, 2003). Collectively, these data have been used to support a model whereby PARP-1 promotes the decondensation of chromatin by causing the dissociation of nucleosomes through (ADP-ribosyl)ation of H1 and core histones, as well as the generation of polyanionic, histone binding PAR (Kim, 2004 and references therein).
A number of aspects of PARP-1 biology have been difficult to reconcile with this model and other prevailing viewpoints in the literature. For example, PARP-1 itself, not H1 or core histones, is the primary protein target for PARP-1-mediated (ADP-ribosyl)ation in vivo, with greater than 90 percent of PAR found on PARP-1. Auto(ADP-ribosyl)ation of PARP-1 has profound effects on PARP-1 activity, including inhibition of PARP-1's DNA binding and enzymatic activities. Furthermore, although PARP-1's enzymatic activity has widely been reported to be strictly dependent on the binding of PARP-1 to damaged DNA, such a requirement would not allow a role for PARP-1 under normal physiological conditions where genome integrity is preserved (Kraus, 2003). In addition to these issues, experimental limitations in some cases, such as the use of PARP-1 contaminated with DNA fragments, chromatin containing free DNA ends resembling double-strand breaks, or assays where the (ADP-ribosyl)ated protein targets were not definitively identified, have made conclusions about PARP-1 function difficult to draw (Kim, 2004 and references therein).
A defined chromatin assembly system containing DNA-free PARP-1 and nick-free circular (i.e., end-free) DNA templates, as well as complementary cell-based assays, was used to examine the effects of PARP-1 on chromatin structure. The results indicate that PARP-1 has specific nucleosome binding properties that can alter Pol II transcription by altering chromatin structure in a manner similar, but not identical, to H1. Unlike H1, however, the activity of PARP-1 is modulated by auto(ADP-ribosyl)ation in the presence of the metabolic cofactor NAD+, adding additional opportunities for regulation. Collectively, these results provide a new framework for thinking about PARP-1 activity in the regulation of chromatin structure and transcription (Kim, 2004).
The incorporation of PARP-1 into chromatin promotes the formation of higher-order chromatin structures that localize to discrete chromatin domains in vivo. As illustrated by immunofluorescent staining of Drosophila polytene chromosomes, these domains are not associated with active Pol II, reflecting the repressive effect that incorporation of PARP-1 has on Pol II transcription in vitro. Of note is the observation that although PARP-1 and H1 share a number of common properties, they reside in distinct (i.e., nonoverlapping) chromatin domains. H1 is localized to highly condensed and transcriptionally repressed chromatin domains, whereas PARP-1 is localized to less-condensed but also transcriptionally repressed chromatin domains that are, perhaps, poised for activation in response to an appropriate signal. Thus, the type of linker DNA binding protein in a particular chromatin domain is likely to set the transcriptional output of the genes contained in that domain. The diverse ways in which PARP-1's activity and association with chromatin can be modulated make PARP-1 an attractive target for regulating dynamic chromatin reconfigurations required for gene repression and activation, as well as other chromatin-based functions (Kim, 2004).
Profound epigenetic differences exist between genomes derived from male and female gametes; however, the nature of these changes remains largely unknown. A systematic investigation was undertaken of chromatin reorganization during gametogenesis, using the model eukaryote Saccharomyces cerevisiae to examine sporulation, which has strong similarities with higher eukaryotic spermatogenesis. A mutational screen of histones H3 and H4 was undertaken to uncover substitutions that reduce sporulation efficiency. Two patches of residues -- one on H3 and a second on H4 -- were discovered that are crucial for sporulation but not critical for mitotic growth, and likely comprise interactive nucleosomal surfaces. Furthermore, novel histone post-translational modifications were discoved that mark the chromatin reorganization process during sporulation. First, phosphorylation of H3T11 appears to be a key modification during meiosis, and requires the meiotic-specific kinase Mek1. Second, H4 undergoes amino tail acetylation at Lys 5, Lys 8, and Lys 12, and these are synergistically important for post-meiotic chromatin compaction, occurring subsequent to the post-meiotic transient peak in phosphorylation at H4S1, and crucial for recruitment of Bdf1, a bromodomain protein, to chromatin in mature spores. Strikingly, the presence and temporal succession of the new H3 and H4 modifications are detected during mouse spermatogenesis, indicating that they are conserved through evolution. Thus, the results show that investigation of gametogenesis in yeast provides novel insights into chromatin dynamics, which are potentially relevant to epigenetic modulation of the mammalian process (Govin, 2010).
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