Histone deacetylase 1
SpHDAC1, a cDNA homolog of the yeast Rpd3 and higher eukaryotic histone deacetylases (HDAC), was
cloned from the sea urchin Strongylocentrotus purpuratus. Its predicted polypeptide and the Rpd3
homologs are highly identical in two-thirds of their lengths, but diverge in their carboxyl-terminal
regions in both length and sequence. SpHDAC1 transcripts, which reach maximal concentration at the
blastula stages, and diminish thereafter, are neither ubiquitously expressed nor restricted to particular
cell lineages, but appear successively in distinct embryonic regions. In the blastula, transcripts are
concentrated in a ring within the vegetal plate, comprising primordial endoderm, and, at the outset of
gastrulation, in primordial hindgut endoderm. However, in early to mid-gastrula transcripts, they also
appear in oral ectoderm. In the late-stage gastrula, expression develops in the foregut. These shifts in
spatial expression, together with an observed developmental blockage by
the histone deacetylase inhibitor trichostatin A, prior to sea urchin gastrulation, suggest a stepwise involvement of SpHDAC1 gene
expression or SpHDAC1 functionality in the events of normal gastrulation (Nemer, 1998).
The C. elegans synthetic multivulva (synMuv) genes act redundantly to antagonize the specification of vulval cell fates, which are promoted by an RTK/Ras pathway. At least 26 synMuv genes have been genetically identified, several of which encode proteins with homologs that act in chromatin remodeling or transcriptional repression. This study reports the molecular characterization of two synMuv genes, lin-37 and lin-54.lin-37 and lin-54 encode proteins in a complex with at least seven synMuv proteins, including LIN-35, the only C. elegans homolog of the mammalian tumor suppressor Rb. Biochemical analyses of mutants suggest that LIN-9, LIN-53, and LIN-54 are required for the stable formation of this complex. This complex is distinct from a second complex of synMuv proteins with a composition similar to that of the mammalian Nucleosome Remodeling and Deacetylase complex. The class B synMuv complex identified in this study is evolutionarily conserved and likely functions in transcriptional repression and developmental regulation (Harrison, 2006; full text of article).
LIN-37 and LIN-54 form a multisubunit protein complex together with at least five other class B synMuv proteins: LIN-9, LIN-35 Rb, LIN-52, LIN-53 RbAp48, and DPL-1 DP. This DP, Rb, and MuvB (DRM) complex is biochemically and genetically distinct from a NuRD-like complex that includes HDA-1 HDAC1, LET-418 Mi2, and LIN-53 RbAp48. These findings suggest that LIN-35 Rb and DPL-1 DP likely have a function in vulval development distinct from recruitment of the NuRD complex (Harrison, 2006).
The DRM complex is similar to two recently described and highly similar complexes that contain several Drosophila homologs of class B synMuv proteins (Korenjak, 2004; Lewis, 2004). The Myb–MuvB complex was purified by immunoprecipitation of the LIN-54 homolog Mip120 or the LIN-9 homolog Mip130 from Drosophila tissue-culture cells and coimmunoprecipitating proteins were identified by mass spectrometry. The Myb–MuvB complex contains stoichiometric levels of Mip130, RBF, Mip40, Mip120, p55, dDP, dE2F2, and dLin52, which are homologs of LIN-9, LIN-35, LIN-37, LIN-54, LIN-53, DPL-1, EFL-1, and LIN-52, respectively. This complex also contains substoichiometric amounts of Rpd3, the fly homolog of HDA-1, and L(3)MBT, a protein similar to the class B synMuv protein LIN-61. The dREAM complex was identified by biochemical purification of Drosophila Rb-containing complexes from embryo extracts followed by mass spectrometry and Western blot analyses. The dREAM complex contains all of the proteins identified in the Myb–MuvB complex at stoichiometric levels except for dLin52 (Korenjak, 2004). The differences between the dREAM and Myb–MuvB complexes might be a consequence of the methods used for purification or might reflect the existence in different tissues or during different developmental stages of multiple subcomplexes with overlapping components. Both the dREAM and Myb–MuvB complexes can mediate transcriptional repression of many E2F-target genes (Harrison, 2006).
The similarity between the C. elegans DRM complex and the Drosophila dREAM and Myb–MuvB complexes indicates that the DRM complex likely also acts in transcriptional repression. Given the broad expression patterns of the synMuv genes and the multiple phenotypic abnormalities caused by the loss of individual synMuv proteins, it is proposed that, similar to their Drosophila counterparts, the DRM complex proteins are involved in the repression of many targets important for diverse biological functions (Harrison, 2006).
The DRM complex differs slightly from both the dREAM and the Myb–MuvB complexes. Unlike the dREAM complex, the DRM complex contains a LIN-52 dLin52-like protein. Unlike the Myb–MuvB complex, the DRM complex does not contain HDA-1 Rpd3 or LIN-61 L(3)MBT. The similarities of the DRM, dREAM, and Myb–MuvB complexes suggest that there is a core complex consisting of LIN-35 RBF, EFL-1 E2F2, DPL-1 DP, LIN-9 Mip130, LIN-37 Mip40, LIN-52 dLin-52, LIN-53 p55, and LIN-54 Mip120 and that this complex might associate with other proteins during specific stages of development or in certain cell types (Harrison, 2006).
The dREAM and Myb–MuvB complexes both contain the DNA-binding protein Myb. There is no clear Myb homolog in C. elegans. It is possible that the C. elegans DRM complex does not contain a Myb ortholog or that the functional ortholog of the Drosophila Myb protein found in the dREAM and Myb–MuvB complexes might not be readily identifiable by comparisons of primary sequence (Harrison, 2006).
It is proposed that the DRM complex could be recruited to DNA by multiple DNA-binding factors, including LIN-54 and the heterodimeric transcription factor formed by EFL-1 and DPL-1. The DRM complex could then act with the NuRD-like complex to repress transcription. Alternatively, the DRM complex and the NuRD-like complex could act sequentially. The NURD-like complex could deacetylate the N-terminal tails of histones, and the DRM subsequently could bind these unmodified histone tails, preventing their acetylation. The dREAM complex previously has been shown to bind unmodified histone H4 tails, supporting this model. This binding might be mediated by LIN-53, because the mammalian homolog RbAp48 binds histone H4. Deacetylated histones are associated with transcriptionally repressed areas of the genome. Thus, by protecting histone tails from future acetylation, the DRM complex could act to maintain transcriptional repression of nearby genes (Harrison, 2006).
Although neither the DRM nor the dREAM complexes contains known chromatin-modifying or chromatin-remodeling enzymes, these complexes might require the activity of a histone deacetylase to mediate transcriptional repression. Mutations in genes encoding components of either the DRM or the NuRD-like complex require an additional class A or class C synMuv mutation to produce a highly penetrant Muv phenotype. However, mutations affecting two of the NuRD-like complex components, HDA-1 and LET-418, alone can cause low penetrance Muv phenotypes, suggesting that the chromatin-remodeling and chromatin-modifying activities of this complex might be required more broadly for the transcriptional repression of genes necessary for proper vulval development than is the activity of the DRM complex. Perhaps other class B synMuv proteins not associated with the DRM complex, for example, HPL-2, LIN-36, or LIN-61, act with the DRM complex to maintain the repressed state formed by the activity of the NuRD-like complex (Harrison, 2006).
The high degree of conservation shared by the DRM/Myb-MuvB/dREAM complexes in C. elegans and Drosophila and the important roles that the components of DRM complex play in C. elegans development suggest that a similar complex exists in other organisms, including humans. The core components of these complexes have homologs in humans, and the human homolog of LIN-9, hLin-9, can associate with Rb to specifically promote differentiation (but not to inhibit cell-cycle progression). Perhaps Rb or other Rb-family proteins act within the context of a DRM-like complex to control differentiation. Rb could act as a tumor suppressor through such DRM-mediated regulation of differentiation in addition to its role in cell-cycle regulation. Further biochemical and genetic studies of nematodes, insects, and mammals should elucidate the role that this conserved protein complex plays in development and in carcinogenesis (Harrison, 2006).
Histone deacetylase in zebrafish In the developing vertebrate retina, progenitor cells initially proliferate
but begin to produce postmitotic neurons when neuronal differentiation occurs.
However, the mechanism that determines whether retinal progenitor cells
continue to proliferate or exit from the cell cycle and differentiate is
largely unknown. Histone deacetylase 1 (Hdac1) is
required for the switch from proliferation to differentiation in the zebrafish
retina. A zebrafish mutant, ascending and descending
(add), was isolated in which retinal cells fail to differentiate into neurons and
glial cells but instead continue to proliferate. The cloning of the
add gene revealed that it encodes Hdac1. Furthermore, the ratio of
the number of differentiating cells to that of proliferating cells increases
in proportion to Hdac activity, suggesting that Hdac proteins regulate a
crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling
promotes the proliferation of retinal cells in zebrafish, and Notch signaling
inhibits neuronal differentiation through the activation of a neurogenic
inhibitor, Hairy/Enhancer-of-split (Hes). It was found that both the Wnt and
Notch/Hes pathways are activated in the add mutant retina. The
cell-cycle progression and the upregulation of Hes expression in the
add mutant retina can be inhibited by the blockade of Wnt and Notch
signaling, respectively. These data suggest that Hdac1 antagonizes these
pathways to promote cell-cycle exit and the subsequent neurogenesis in
zebrafish retina. Taken together, these data suggest that Hdac1 functions as a
dual switch that suppresses both cell-cycle progression and inhibition of
neurogenesis in the zebrafish retina (Yamaguchi, 2005).
Vertebrate gastrulation involves the coordinated movements of populations of cells. These movements include cellular rearrangements in which cells polarize along their medio-lateral axes leading to cell intercalations that result in elongation of the body axis. Molecular analysis of this process has implicated the non-canonical Wnt/Frizzled signaling pathway that is similar to the planar cell polarity pathway (PCP) in Drosophila. This study describes a zebrafish mutant, colgate (col), which displays defects in the extension of the body axis and the migration of branchiomotor neurons. Activation of the non-canonical Wnt/PCP pathway in these mutant embryos by overexpressing ΔNdishevelled, rho kinase2 and van gogh-like protein 2 (vangl2) rescues the extension defects suggesting that col acts as a positive regulator of the non-canonical Wnt/PCP pathway. Further, col is shown to normally regulate the caudal migration of nVII facial hindbrain branchiomotor neurons; the mutant phenotype can be rescued by misexpression of vangl2 independent of the Wnt/PCP pathway. col locus was cloned and found to encode histone deacetylase1 (hdac1). hdac1 has been implicated in repressing the canonical Wnt pathway. This study demonstrates novel roles for zebrafish hdac1 in activating non-canonical Wnt/PCP signaling underlying axial extension and in promoting Wnt-independent caudal migration of a subset of hindbrain branchiomotor neurons (Nambiar, 2007).
Studies of col mutants have revealed novel functions of Hdac1 in major signaling pathways regulating embryonic development. However, precisely how Hdac1 functions in these pathways is not fully understood. In the canonical Wnt pathway, Hdac1 functions as a co-repressor with molecules such as Groucho and LEF1 in the nucleus. Studies in Drosophila and vertebrates have shown that Groucho, a canonical Wnt signaling pathway repressor, readily interacts with Hdac1 forming a repressor complex that remains tethered to the promoter of Wnt target genes. Data also indicates that the Wnt transcription factor LEF1 can act as a repressor in the presence of Hdac1. Activation of LEF-dependent target genes occurs when the increasing level of β-catenin in the nucleus is able to dissociate Hdac1 from LEF1 and itself bind to LEF1 to form a dimeric activator. Thus, Hdac1 appears to maintain Wnt target genes in a repressed state until replaced by activators such as β-catenin (Nambiar, 2007).
This study has shown that col/hdac1 regulates both the non-canonical Wnt/PCP pathway that controls CE movements as well as the pathway that mediates the caudal migration of hindbrain facial motor neurons. There are a number of possible ways in which Hdac1 functions in these pathways. For example, since Hdac1 regulates both pathways, it is conceivable then that Col/Hdac1 could act by regulating the transcription of vangl2 or its interacting proteins. vangl2 expression was examined in col mutants and there appeared to be no significant difference compared to wildtype siblings. Another possible scenario for the functioning of Col/Hdac1 in this context could be via an interaction with Vangl2 and its interacting proteins such as Pk and Scribble that act at the common branchpoint. Another possibility is that Col/Hdac1 regulates the transcription of other components of the Wnt/PCP pathway and/or the targets of Wnt/PCP pathway genes. In the latter case, additional interactions of Hdac1 with Wnt/PCP signaling-independent genes or components of the pathway that also regulate branchiomotor neuron migration are possible. Further studies exploring the function of col should reveal the molecular mechanism by which col/hdac1 affects the activities of the genes involved in the morphogenetic events that were described (Nambiar, 2007).
Histone deacetylase in Xenopus HDm is a
maternally expressed putative deposition histone deacetylase that has been cloned from Xenopus laevis. Comparison of the
amino acid sequences of histone deacetylases from diverse eukaryotes shows high levels of identity
within a putative enzyme core region. There is also alignment with other types of protein: acetoin-utilizing
enzymes from eubacteria; acetylpolyamine hydrolases from mycoplasma and cyanobacteria, and a protein
of unknown function from an archaebacterium, all of which reveal an apparently conserved core. This suggests that
histone deacetylases belong to an ancient family of enzymes with related functions (Ladomery, 1997).
A multi-subunit
complex has been purified from Xenopus laevis eggs which contains six putative subunits including the known deacetylase
subunits Rpd3 and RbAp48/p46 as well as substoichiometric quantities of the deacetylase-associated
protein Sin3. In addition, one of the other components of the complex has been identified as Mi-2 (see Drosophila Mi-2), a
Snf2 superfamily member previously identified as an autoantigen in the human connective tissue disease
dermatomyositis. Mi-2's Drosophila homolog physically interacts with Drosophila Hunchback and is involved in Hunchback mediated repression (Kehle, 1998). Nucleosome-stimulated ATPase activity precisely copurifies with
both histone deacetylase activity and the deacetylase enzyme complex. This association of a histone
deacetylase with a Snf2 superfamily ATPase suggests a functional link between these two disparate
classes of chromatin regulators (Wade, 1998).
Histone deacetylase and chromatin assembly contribute to the control of transcription of the Xenopus
TRbetaA gene promoter by the heterodimer of Xenopus thyroid hormone receptor and 9-cis retinoic acid
receptor (TR-RXR). Addition of the histone deacetylase inhibitor Trichostatin A (TSA) relieves repression
of transcription due to chromatin assembly following microinjection of templates into Xenopus oocyte
nuclei, and eliminates regulation of transcription by TR-RXR. Expression of Xenopus RPD3p, the
catalytic subunit of histone deacetylase, represses the TRbetaA promoter, but only after efficient
assembly of the template into nucleosomes. In contrast, the unliganded TR-RXR represses templates only
partially assembled into nucleosomes; addition of TSA also relieves this transcriptional repression. This
result indicates the distinct requirements for chromatin assembly in mediating transcriptional repression
by the deacetylase alone, compared with those needed in the presence of unliganded TR-RXR. In
addition, whereas hormone-bound TR-RXR targets chromatin disruption as assayed through changes in
minichromosome topology and loss of a regular nucleosomal ladder on micrococcal nuclease digestion,
addition of TSA relieves transcriptional repression but does not disrupt chromatin. Thus, TR-RXR can
facilitate transcriptional repression in the absence of hormone through mechanisms in addition to
recruitment of deacetylase, and disrupt chromatin structure through mechanisms in addition to the
inhibition or release of deacetylase (Wong, 1998).
The protein associations and enzymatic requirements were investigated for the
Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional
repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and
cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear.
Xenopus RbAp48 and SIN3 have been cloned and it has been shown that expression of RPD3, but not RbAp48 or
SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase
activity and transcriptional repression of the TRbetaA promoter. This repression
requires deacetylase activity and nuclear import of RPD3 mediated by a
carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated
into oocyte deacetylase and ATPase complexes but
cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus.
RPD3 associates with RbAp48 through N- and C-terminal contacts and
RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the
segment of the N-terminal tail immediately proximal to the histone fold domain
of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through
interaction with endogenous RbAp48 to direct transcriptional repression of the
Xenopus TRbetaA promoter in the oocyte nucleus. However, the exogenous RPD3
deacetylase functions to repress transcription in the absence of a requirement
for association with SIN3 or other targeted corepressors (Vermaak, 1999).
Trapoxin is a microbially derived cyclotetrapeptide that inhibits histone deacetylation in vivo and causes mammalian cells to arrest in the cell cycle. A trapoxin affinity
matrix was used to isolate two nuclear proteins that copurify with histone deacetylase activity. Both proteins were identified by peptide microsequencing, and a
complementary DNA encoding the histone deacetylase catalytic subunit (HD1) was cloned from a human Jurkat T cell library. As the predicted protein is very
similar to the yeast transcriptional regulator Rpd3p, these results support a role for histone deacetylase as a key regulator of eukaryotic transcription (Taunton, 1995).
The histone deacetylase domain of almost all members of higher eukaryotic histone deacetylases already
identified (HDAC family) is highly homologous to that of yeast RPD3. Two cDNAs encoding members of a new family of histone deacetylase in mouse have been cloned that show an even better
homology to yeast HDA1 histone deacetylase. These cDNAs encode relatively large proteins, presenting
an in vitro trichostatin A-sensitive histone deacetylase activity. Interestingly, one, mHDA2, encodes a
protein with two putative deacetylase domains, and the other, mHDA1, contains only one deacetylase
homology domain, located at the C-terminal half of the protein. These newly
identified genes could belong to a network of genes coordinately regulated and involved in the remodeling
of chromatin during cell differentiation. Indeed, the expression of mHDA1 and mHDA2 is tightly linked to
the state of cell differentiation, behaving therefore like the histone H1 degrees-encoding gene. Moreover,
like histone H1(0) gene, mHDA1 and mHDA2 gene expression is induced upon deacetylase inhibitor
treatment. The existence of a regulatory mechanism is postulated, one commanding a coordinate expression of
a group of genes involved in the remodeling of chromatin not only during cell differentiation but also after
abnormal histone acetylation (Verdel, 1999).
A human ortholog of RPD3, HDAC3, has been identifed. This cDNA encodes a protein of 428 amino acids with
58% sequence identity with HDAC1p. By using a specific polyclonal antiserum recognizing the C-terminal
domain of HDAC3p and Western blotting, a single approximately 49-kDa band was identifed in several
tumor cell lines. HDAC3p is expressed predominantly in the nuclear compartment. Immunoprecipitation
experiments with either an antiserum against HDAC3p or an anti-FLAG antiserum and a flagged HDAC3
cDNA show that HDAc3p exhibits deacetylase activity both on free histones and on purified
nucleosomes. This deacetylase activity is inhibited by trichostatin, trapoxin, and butyrate in vitro to the
same degree as the deacetylase activity associated with HDAC1p. These observations identify another
member of a growing family of human HDAC genes (Emiliani, 1998).
Gene expression is in part controlled by chromatin remodeling factors and the acetylation state of
nucleosomal histones. The latter process is regulated by histone acetyltransferases and histone
deacetylases (HDACs). Previously, three human and five yeast HDAC enzymes have been identified. These
can be categorized into two classes: the first class represented by yeast Rpd3-like proteins and the second
by yeast Hda1-like proteins. Human HDAC1, HDAC2, and HDAC3 proteins are members of the first
class, whereas no class II human HDAC proteins have been previously identified. The amino acid sequence of Hda1p
was used to search the GenBank/expressed sequence tag databases to identify partial sequences from
three putative class II human HDAC proteins. The corresponding full-length cDNAs were cloned and
defined as HDAC4, HDAC5, and HDAC6. These proteins possess certain features present in the
conserved catalytic domains of class I human HDACs, but also contain additional sequence domains.
Interestingly, HDAC6 contains an internal duplication of two catalytic domains, which appear to function
independently of each other. These class II HDAC proteins have differential mRNA expression in human
tissues and possess in vitro HDAC activity that is inhibited by trichostatin A. Coimmunoprecipitation
experiments indicate that these HDAC proteins are not components of the previously identified HDAC1
and HDAC2 NRD and mSin3A complexes. However, HDAC4 and HDAC5 associate with HDAC3 in vivo.
This finding suggests that the human class II HDAC enzymes may function in cellular processes distinct
from those of HDAC1 and HDAC2 (Grozinger, 1999).
Recently cloned and characterized has been a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone
deacetylases. Analysis of the predicted amino acid sequence of HDAC-A reveals an open reading frame
of 967 amino acids containing two domains: a NH2-terminal domain with no homology to known proteins
and a COOH-terminal domain with homology to known histone deacetylases (42% similarity to RPD3,
60% similarity to HDA1). Three additional human cDNAs with high homology to HDAC-A have been identified
in sequence data bases, indicating that HDAC-A itself is a member of a new family of human histone
deacetylases. The mRNA encoding HDAC-A is differentially expressed in a variety of human tissues.
The expressed protein, HDAC-Ap, exhibits histone deacetylase activity and this activity maps to the
COOH-terminal region (amino acids 495-967) with homology to HDA1p. In immunoprecipitation
experiments, HDAC-A interacts specifically with several cellular proteins, indicating that it might be part
of a larger multiprotein complex (Fischle, 1999).
Reversible acetylation of histone proteins plays a critical role in transcriptional regulation, cell cycle
progression, and developmental events. The steady state of histone acetylation is controlled by the
enzymatic activities of multiple histone acetyltransferases and histone deacetylases (HDACs). Three
distinct human HDACs are homologous to RPD3, a yeast transcriptional regulator. A genomic clone for the human HDAC3 gene has been isolated and
sequenced. This is a single-copy gene spanning a region of
at least 13 kb. Determination of the intron-exon splice junctions has established that the gene is encoded by 15
exons ranging in size from 56 bp to 657 bp. Fluorescence in situ hybridization studies have localized this gene to
5q31. Double-target experiments, in which both HDAC3 and the early-growth response 1 gene (EGR1,
which is localized in the 5q31.2 region) were used as probes, show that the HDAC3 gene lies in region
5q31.3, immediately distal to EGR1 with respect to the centromere (Mahlknecht, 1999).
The Mi-2 complex has been implicated in chromatin remodeling and transcriptional
repression associated with histone deacetylation. A purified Mi-2
complex containing six components (Mi-2, Mta 1-like, p66, RbAp48, RPD3, and
MBD3) has been used to investigate the capacity of this complex to destabilize histone-DNA
interactions and deacetylate core histones. The Mi-2 complex has ATPase activity
that is stimulated by nucleosomes but not by free histones or DNA. This
nucleosomal ATPase is relatively inefficient, yet is essential to facilitate
both translational movement of histone octamers relative to DNA and the
efficient deacetylation of the core histones within a mononucleosome.
Surprisingly, ATPase activity has no effect on deacetylation of nucleosomal
arrays (Guschin, 2000).
MBD2 and MBD3 (see Drosophila MBD-like) are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription (Jin, 2005).
Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from lysine residues of histone and nonhistone proteins. Recent studies suggest that they are key regulators of many cellular events, including cell proliferation and cancer development. Human class I HDACs possess homology to the yeast RPD3 protein and include HDAC1, HDAC2, HDAC3, and HDAC8. While HDAC1, HDAC2, and HDAC3 have been characterized extensively, almost nothing is known about HDAC8. HDAC8 is phosphorylated by cyclic AMP-dependent protein kinase A (PKA) in vitro and in vivo. The PKA phosphoacceptor site of HDAC8 is Ser(39), a nonconserved residue among class I HDACs. Mutation of Ser(39) to Ala enhances the deacetylase activity of HDAC8. In contrast, mutation of Ser(39) to Glu or induction of HDAC8 phosphorylation by forskolin, a potent activator of adenyl cyclase, decreases HDAC8's enzymatic activity. Remarkably, inhibition of HDAC8 activity by hyperphosphorylation leads to hyperacetylation of histones H3 and H4, suggesting that PKA-mediated phosphorylation of HDAC8 plays a central role in the overall acetylation status of histones (Lee, 2004).
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor
composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator
(ARNT/HIF1ß). ARNT function is required
for murine placental development. Cultured trophoblast stem (TS)
cells were used to investigate the molecular basis of this requirement. In
vitro, wild-type TS cell differentiation is largely restricted to
spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS
cells differentiate into chorionic trophoblasts and syncytiotrophoblasts, as
demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the
HIV receptor CXCR4. During this process, a region of the differentiating
Arnt-null TS cells undergo granzyme B-mediated apoptosis,
suggesting a role for this pathway in murine syncytiotrophoblast turnover.
Surprisingly, HIF1alpha and HIF2alpha are induced during TS cell
differentiation in 20% O2; additionally, pVHL levels are modulated
during the same time period. These results suggest that oxygen-independent HIF
functions are crucial to this differentiation process. Since histone deacetylase
(HDAC) activity has been linked to HIF-dependent gene expression,
whether ARNT deficiency affects this epigenetic regulator was investigated.
Interestingly, Arnt-null TS cells have reduced HDAC activity,
increased global histone acetylation, and altered class II HDAC subcellular
localization. In wild-type TS cells, inhibition of HDAC activity recapitulates
the Arnt-null phenotype, suggesting that crosstalk between the HIFs
and the HDACs is required for normal trophoblast differentiation. Thus, the
HIFs play important roles in modulating the developmental plasticity of stem
cells by integrating physiological, transcriptional and epigenetic inputs (Maltepe, 2005).
Ski was first identified as a viral oncogene (v-ski) from the avian Sloan-Kettering retrovirus (SKV) that transforms chicken embryo fibroblasts. The human cellular homolog c-ski was later cloned based on its homology with v-ski and was found to encode a nuclear protein of 728 amino acids. Compared with c-Ski, v-Ski is truncated mostly at the carboxyl terminus. However, this truncation is not responsible for the activation of ski as an oncogene. Overexpression of wild-type c-Ski also results in oncogenic transformation of chicken and quail embryo fibroblasts. The transforming activity of Ski is likely attributable to overexpression, not truncation, of the c-Ski protein. Consistent with this notion, an elevated level of c-Ski has been detected in several human tumor cell lines derived from neuroblastoma, melanoma, and prostate cancer. c-ski is a unique oncogene; in addition to affecting cell growth, it is also involved in regulation of muscle differentiation. Overexpression of Ski results in muscle differentiation of quail embryo cells and hypertrophy of skeletal muscle in mice. Furthermore, mice lacking c-ski display defective muscle and neuronal differentiation.
At the molecular level, Ski can function either as a transcriptional activator or as a repressor depending on the specific promoters involved. It has been shown to bind to DNA, but only in conjunction with other cellular proteins. Ski is a component of the histone deacetylase (HDAC1) complex through binding to the nuclear hormone receptor corepressor (N-CoR) and mSin3A, and mediated transcriptional repression of the thyroid hormone receptor, Mad and pRb. The interaction between Ski and N-CoR is mediated by the amino-terminal part of Ski. This region is also essential for the transforming activity of c-Ski and is conserved among ski family members, including v-Ski and c-SnoN. This raises an interesting possibility that the transforming activity of Ski may be linked to its function as a transcriptional corepressor (Luo, 1999 and references therein).
Ski can interact directly with Smad2, Smad3, and Smad4 on a TGF beta-responsive promoter element and repress their abilities to activate transcription through
recruitment of the nuclear transcriptional corepressor N-CoR, and
possibly its associated histone deacetylase complex. Thus Ski is a transcriptional corepressor of Smads. Overexpression of
Ski in a TGF beta-responsive cell line renders it resistant to
TGF beta-induced growth inhibition and defective in activation of JunB
expression. This ability to overcome TGF beta-induced growth arrest may
be responsible for the transforming activity of Ski in human and avian
cancer cells. These studies suggest a new paradigm for inactivation of
the Smad proteins by an oncoprotein through transcriptional repression (Luo, 1999).
Using a nuclear extract from c-ski-transformed cells, a specific DNA-binding site for Ski and its associated proteins was identified (GTCTAGAC) by cyclic amplification and selection of targets (CASTing). The Ski binding site was found to mediate transcriptional repression by Ski, suggesting that Ski may bind to DNA through interaction with the Smads. Ski/Smad3 and Ski/Smad4 complexes can bind to SBE and repress Smad-mediated transcriptional activation. Thus, Smad3 and Smad4 are the DNA-binding partners of Ski in these c-ski-transformed cells. In addition to SBE, Ski has also been found to interact with the nuclear factor I (NFI) binding site through interaction with the NFI protein. However, in this context, Ski functions to potentiate, not repress, NFI-stimulated transcriptional activation. Thus, Ski may interact with different DNA-binding factors and regulate transcription both positively and negatively depending on the proper cellular context or interacting partners (Luo, 1999 and references).
Ski also binds directly to Rb and retinoic acid receptor and to repress transactivation induced by these proteins, probably through similar mechanisms. N-CoR was originally identified as a corepressor that mediates transcriptional repression by the thyroid hormone receptor and Mad. It is a protein of 270 kD and contains three repressor domains in its amino-terminal region. It shows a striking homology to another corepressor, SMRT, and represses transcription by forming complexes with mSin3 and HDAC. Although no specific interactions between the Smads and endogenous mSin3A or HDAC could be detected because of technical difficulties, the recruitment of an N-CoR complex to the Smads suggests that repression of Smad-mediated transcription by Ski may involve deacetylation of nucleosomal histones. Recently, Smad2 has been shown to interact with TGIF, another transcriptional corepressor that recruits HDAC to the Smads. Thus, repression of Smad-mediated transactivation may involve multiple corepressors. Future studies will allow for a determination of whether Ski, Smads, N-CoR, and TGIF are in the same complex or whether Smads interact with different corepressors depending on the expression level of these corepressors in different cell types or at different developmental stages (Luo, 1999 and references therein).
Smads are intracellular signaling mediators of the transforming growth factor-β superfamily that regulates a wide variety of biological processes. Among them, Smads 2 and 3 are activated specifically by TGF-β. c-Ski has been identified as a Smad2 interacting protein. c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-β-dependent manner. c-Ski contains two distinct Smad-binding sites with different binding properties. c-Ski strongly inhibits transactivation of various reporter genes by TGF-β. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3 with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-β signaling
(Akiyoshi, 1999).
Transcriptional repression by nuclear receptors has been correlated to binding of the putative
co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive
co-repressor mSin3, and the histone deacetylase mRPD3: this complex is required for both nuclear
receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain
family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from
repressor to activator functions involves the exchange of complexes containing histone deacetylases with
those that have histone acetylase activity (Heinzel, 1997).
Sin3 and Rpd3 negatively regulate a diverse set of yeast genes. A mouse Sin3-related protein is a
transcriptional corepressor, and a human Rpd3 homolog is a histone deacetylase.
Sin3 and Rpd3 are specifically required for transcriptional repression by Ume6, a DNA-binding protein
that regulates genes involved in meiosis. A short region of Ume6 is sufficient to repress transcription, and
this repression domain mediates a two-hybrid and physical interaction with Sin3. Coimmunoprecipitation
and two-hybrid experiments indicate that Sin3 and Rpd3 are associated in a complex distinct from TFIID
and Pol II holoenzyme. Rpd3 is specifically required for repression by Sin3, and artificial recruitment of
Rpd3 results in repression. These results suggest that repression by Ume6 involves recruitment of a
Sin3-Rpd3 complex and targeted histone deacetylation (Kadosh, 1997).
A recently identified mammalian histone
deacetylase (HD1) shows homology to the yeast Rpd3 protein, which together with Sin3 affects the
transcription of several genes. Mammalian Sin3 proteins interact with the Mad components of the
Myc/Max/Mad network of cell growth regulators. Mad/Max complexes may recruit mammalian Rpd3-like
enzymes, thus directing histone deacetylase activity to promoters and negatively regulating cell
growth. A tetrameric complex composed of Max, Mad1, Sin3B
and HD1 is reported. This complex has histone deacetylase activity that can be blocked by the histone deacetylase
inhibitors trichostatin A and sodium butyrate. The inhibition of cell growth by Mad1 is enhanced by Sin3B
and HD1, as measured by colony formation assays. Furthermore, a Mad1-induced block of S-phase
progression can be overcome by trichostatin A, as shown in microinjection experiments.
It is concluded that the recruitment of a histone deacetylase by sequence-specific DNA-binding proteins
provides a mechanism by which the state of acetylation of histones in nucleosomes and hence the activity
of specific promoters can be influenced. The finding that Mad/Max complexes interact with Sin3 and
HD1 in vivo suggests a model for the role of Mad proteins in antagonizing the function of Myc proteins (Sommer, 1997).
Thyroid hormone and retinoic acid receptors are members of the nuclear receptor superfamily of
ligand-dependent transcription factors that stimulate the transcription of target genes in the presence of
activating ligands and repress transcription in their absence. Transcriptional repression by the thyroid
hormone and retinoic acid receptors has been proposed to be mediated by the nuclear receptor
corepressor, N-CoR, or the related factor, SMRT (silencing mediator of retinoic acid and thyroid
hormone receptors). Recent studies have suggested that transcriptional repression by N-CoR involves a
corepressor complex that also contains mSin3A/B and the histone deacetylase, RPD3. Transcriptional repression by the retinoic acid receptor can be either positively or
negatively regulated by changes in the levels of N-CoR expression, suggesting a relatively strict
stoichiometric relationship between N-CoR and other components of the corepressor complex. Consistent
with this interpretation, overexpression of several functionally defined domains of N-CoR also relieve
repression by nuclear receptors. N-CoR is distributed throughout the nucleus in a nonuniform pattern,
and a subpopulation becomes concentrated into several discrete dot structures when highly expressed.
RPD3 is also widely distributed throughout the nucleus in a nonuniform pattern. Simultaneous imaging of
RPD3 and N-CoR suggest that a subset of each of these proteins colocalize, consistent with the existence
of coactivator complexes containing both proteins. In addition, a substantial fraction of both N-CoR and
mSin3 A/B appear to be independently distributed. These observations suggest that interactions between
RPD3 and Sin3/N-CoR complexes may be dynamically regulated (Soderstrom, 1997).
The retinoblastoma protein (RB) facilitates adipocyte differentiation by inducing cell cycle arrest and enhancing the transactivation by the adipogenic CCAAT/enhancer binding proteins (C/EBP). The peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor pivotal for adipogenesis, promotes adipocyte differentiation more efficiently in the absence of RB. PPARgamma and RB coimmunoprecipitate, and this PPARgamma-RB complex also contains the histone deacetylase HDAC3, thereby attenuating PPARgamma's capacity to drive gene expression and adipocyte differentiation. Dissociation of the PPARgamma-RB-HDAC3 complex by RB phosphorylation or by inhibition of HDAC activity stimulates adipocyte differentiation. These observations underscore an important function of both RB and HDAC3 in fine-tuning PPARgamma activity and adipocyte differentiation (Fajas, 2002).
The detection of a PPARgamma-RB-HDAC3 protein complex on the promoter of well-established PPARgamma targets in vivo, as well as the fact that the proadipogenic effects of PPARgamma are blunted in one case by the presence of RB and in the other, stimulated by HDAC inhibitors, underscores the relevance of the repression of PPARgamma by RB and HDAC3. Complexes involved in nuclear receptor-mediated transcriptional repression often contain HDACs. The thyroid receptor, as does PPARgamma, specifically recruits HDAC3. Participation of HDACs in the repressive activity of nuclear receptor is facilitated by the presence of other corepressors, such as the silencing mediator of retinoic and thyroid receptors (SMRT) or the nuclear receptor corepressor (N-CoR). Furthermore, corepressors such as SMRT and N-CoR are required for the activation of HDAC3 in such complexes. It is therefore tempting to speculate that RB fulfills such a corepressor role for PPARgamma (Fijas, 2002).
This study investigated recruitment of coactivators (SRC-1, SRC-2, and SRC-3) and corepressors (HDAC1, HDAC2, HDAC3, SMRT, and NCoR) to the IkappaBα gene promoter after NF-kappaB activation by tumor necrosis factor-α. The data from chromatin immunoprecipitation assay suggest that coactivators and corepressors are simultaneously recruited to the promoter, and their binding to the promoter DNA is oscillated in HEK293 cells. SRC-1, SRC-2, and SRC-3 all enhanced IkappaBα transcription. However, the interaction of each coactivator with the promoter exhibited different patterns. After tumor necrosis factor-α treatment, SRC-1 signal was increased gradually, but SRC-2 signal was reduced immediately, suggesting replacement of SRC-2 by SRC-1. SRC-3 signal was increased at 30 min, reduced at 60 min, and then increased again at 120 min, suggesting an oscillation of SRC-3. The corepressors were recruited to the promoter together with the coactivators. The binding pattern suggests that the corepressor proteins formed two types of corepressor complexes, SMRT-HDAC1 and NCoR-HDAC3. The two complexes exhibited a switch at 30 and 60 min. The functions of cofactors were confirmed by gene overexpression and RNA interference-mediated gene knockdown. These data suggest that gene transactivation by the transcription factor NF-kappaB is subject to the regulation of a dynamic balance between the coactivators and corepressors. This model may represent a mechanism for integration of extracellular signals into a precise control of gene transcription (Gao, 2005).
Understanding how dopamine (DA) phenotypes are acquired in midbrain DA (mDA) neuron development is important for bioassays and cell replacement therapy for mDA neuron-associated disorders. This study demonstrate a feed-forward mechanism of mDA neuron development involving Nurr1 (Drosophila homolog Hormone receptor-like in 38) and Foxa2. Nurr1 acts as a transcription factor for DA phenotype gene expression. However, Nurr1-mediated DA gene expression was inactivated by forming a protein complex with CoREST, and then recruiting histone deacetylase 1 (Hdac1), an enzyme catalyzing histone deacetylation, to DA gene promoters. Co-expression of Nurr1 and Foxa2 was established in mDA neuron precursor cells by a positive cross-regulatory loop. In the presence of Foxa2, the Nurr1-CoREST interaction was diminished (by competitive formation of the Nurr1-Foxa2 activator complex), and CoREST-Hdac1 proteins were less enriched in DA gene promoters. Consequently, histone 3 acetylation (H3Ac), which is responsible for open chromatin structures, was strikingly increased at DA phenotype gene promoters. These data establish the interplay of Nurr1 and Foxa2 as the crucial determinant for DA phenotype acquisition during mDA neuron development (Yi 2014).
Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle
progression, and developmental events. The acetylation state of histones is controlled by the activities of
acetylating and deacetylating enzymes. By using differential mRNA display, a mouse
histone deacetylase gene, HD1, has been identified as an interleukin-2-inducible gene in murine T cells. Sequence alignments
reveal that murine HD1 is highly homologous to the yeast RPD3 pleiotropic transcriptional regulator.
Mouse HD1 is a nuclear protein. When expressed
in yeast, murine HD1 is also detected in the nucleus, although it fails to complement the rpd3delta
deletion phenotype. HD1 mRNA expression is low in G0 mouse cells but increases when the cells
crosses the G1/S boundary after growth stimulation. Immunoprecipitation experiments and functional in
vitro assays show that HD1 protein is associated with histone deacetylase activity. Both HD1 protein
levels and total histone deacetylase activity increases upon interleukin-2 stimulation of resting B6.1 cells.
When coexpressed with a luciferase reporter construct, HD1 acts as a negative regulator of the Rous
sarcoma virus enhancer/promoter. HD1 overexpression in stably transfected Swiss 3T3 cells causes a
severe delay during the G2/M phases of the cell cycle. These results indicate that balanced histone
acetylation/deacetylation is crucial for normal cell cycle progression of mammalian cells (Bartl, 1997).
Histone deacetylases (HDACs) modulate chromatin structure and transcription, but little is known about their function in mammalian development. Previously, HDACs have been shown to be required for embryonic development of invertebrates. In addition, loss of specific components of the Sin3 and the NuRD complexes such as RbAp46/48 (lin-53, rba-1), Sin3 (dSin3A), Mi-2 (dMi-2, chd-3, chd-4) and
MTA1/MTA2 (egl-27, egr1) affect embryonic viability and development of Drosophila melanogaster and Caenorhabditis elegans. Mammalian HDAC1 has been implicated in the repression of genes required for cell proliferation and differentiation. Targeted disruption of both HDAC1 alleles results in embryonic lethality before E10.5 due to severe proliferation defects and retardation in development. HDAC1-deficient embryonic stem cells show reduced proliferation rates, which correlate with decreased
cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1. Similarly, expression of p21 and p27 is up-regulated in HDAC1-null embryos. In addition, loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 and concomitant changes in other histone modifications. The expression of HDAC2 and
HDAC3 is induced in HDAC1-deficient cells, but cannot compensate for loss of the enzyme, suggesting a unique function for HDAC1. This study provides the first
evidence that a histone deacetylase is essential for unrestricted cell proliferation by repressing the expression of selective cell cycle inhibitors (Lagger, 2002).
HDAC1-deficient embryos and HDAC1-null cells show proliferation defects. Together with previous data showing high expression levels of
HDAC1 in proliferating cells, these results are suggestive of a proliferation-linked function for the enzyme. Paradoxically, the recruitment of class I
HDACs by Rb seems to be important for the repression of proliferation-associated genes, and HDAC1 should therefore act rather as a growth inhibitor. However, the data shown in this report demonstrate that one of the key functions of HDAC1 is to prevent the expression of antiproliferative genes in cycling cells. These findings indicate that deacetylases other than HDAC1 as well as deacetylase-independent mechanisms
ensure the proper regulation of Rb target genes in HDAC1-null cells. Evidence has been presented that HDAC1 controls the expression of a specific subset of CDK inhibitors. The induction of p21 and p27 in HDAC1-null cells
correlates with the hyperacetylation of the corresponding promoters. The specificity of this response is underlined by the fact that only the proximal but not the distal
portion of the p21 promoter was found to be associated with hyperacetylated histone H3. The proximal p21 promoter contains Sp1-binding sites that are required
for the induction of the p21 gene by deacetylase inhibitors. Activation of tumor suppressors has been shown to be a crucial function of HDAC
inhibitors as anti-cancer drugs in human cells. These results strongly support the idea that HDAC1 might be a relevant target in tumor treatment (Lagger, 2002).
Histone deacetylases (HDACs) regulate gene expression by deacetylating histones and also modulate the acetylation of a number of nonhistone proteins, thus impinging on various cellular processes. This study analyzed the major class I enzymes HDAC1 and HDAC2 in primary mouse fibroblasts and in the B-cell lineage. Fibroblasts lacking both enzymes fail to proliferate in culture and exhibit a strong cell cycle block in the G1 phase that is associated with up-regulation of the CDK inhibitors p21(WAF1/CIP1) and p57(Kip2) and of the corresponding mRNAs. This regulation is direct, as in wild-type cells HDAC1 and HDAC2 are bound to the promoter regions of the p21 and p57 genes. Furthermore, analysis of the transcriptome and of histone modifications in mutant cells demonstrated that HDAC1 and HDAC2 have only partly overlapping roles. Next, HDAC1 and HDAC2 were eliminated in the B cells of conditionally targeted mice. It was found that B-cell development strictly requires the presence of at least one of these enzymes: When both enzymes are ablated, B-cell development is blocked at an early stage, and the rare remaining pre-B cells show a block in G1 accompanied by the induction of apoptosis. In contrast, elimination of HDAC1 and HDAC2 in mature resting B cells has no negative impact, unless these cells are induced to proliferate. These results indicate that HDAC1 and HDAC2, by normally repressing the expression of p21 and p57, regulate the G1-to-S-phase transition of the cell cycle (Yamaguchi, 2010).
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