Enhancer of bithorax/NURF301


The monoclonal antibody Alz50 recognizes both neurofibrillary pathology associated with Alzheimer's disease and subplate neurons in the developing human brain. To attempt to identify Alz50 antigens expressed during development, a human fetal brain cDNA library was immunoscreened. A positive clone was isolated and sequenced. The clone represents a novel gene named FAC1 (Fetal Alz-50-Reactive Clone 1). The FAC1 gene is located on human chromosome 17 and is conserved across species. In the human fetal brain, the FAC1 gene product is abundantly expressed and the protein is located both in the nucleus and the cytoplasm of cells throughout the developing cortex. Decreased levels of FAC1 protein are observed in adult brain by immunoblot analysis. By immunocytochemistry, the FAC1 protein is almost exclusively localized in the nucleus of neurons in the adult neocortex. Therefore, expression of the FAC1 gene is developmentally regulated and the cellular localization of the protein product is altered during development (Bowser, 1995).

Fetal Alz-50 clone 1 (FAC1) is a novel DNA binding protein with altered expression and subcellular localization during neuronal development and degeneration. FAC1 localizes to the cell body and neurites in undifferentiated neurons during development and in degenerating neurons during Alzheimer's disease progression. In the normal adult brain FAC1 is present predominantly in the nucleus of cortical neurons. When in the nucleus, FAC1 has been shown to repress transcription by binding a specific DNA sequence. The affinity of FAC1 for the identified DNA sequence is dramatically enhanced when FAC1 is phosphorylated. Phosphatase treatment of neuroblastoma nuclear extracts reduces FAC1 DNA binding affinity. Finally, inhibition of cellular serine/threonine phosphatases results in increased FAC1 DNA binding activity. These data suggest that FAC1 DNA binding activity is dependent upon and regulated by phosphorylation signals in the cell (Jordan-Sciutto. 1999a).

Fetal Alz-50 clone 1 (FAC1) is a novel, developmentally regulated gene that exhibits changes in protein expression and subcellular localization during neuronal development and neurodegeneration. To understand the functional implications of altered subcellular localization, a normal cellular function of FAC1 has been established. The FAC1 amino acid sequence contains regional homology to transcriptional regulators. Using the polymerase chain reaction-assisted binding site selection assay, a DNA sequence recognized by recombinant FAC1 has been identified. Mutation of any 2 adjacent base pairs in the identified binding site dramatically reduces the binding preference of FAC1, demonstrating that the binding is specific for the identified site. Nuclear extracts from neural and non-neural cell lines contain a DNA-binding activity with similar specificity and nucleotide requirements as the recombinant FAC1 protein. This DNA-binding activity can be attributed to FAC1 since it is dependent upon the presence of FAC1 and behaves identically on a nondenaturing polyacrylamide gel as transiently transfected FAC1. In NIH3T3 cells, luciferase reporter plasmids containing the identified binding site (CACAACAC) are repressed by cotransfected FAC1 whether the binding site is proximal or distal to the transcription initiation site. This study indicates that FAC1 is a DNA-binding protein that functions as a transcription factor when localized to the nucleus (Jordan-Sciutto, 1999b).

The bromodomain is a 110-amino-acid conserved structural region associated with proteins that regulate signal-dependent, nonbasal transcription. The bromodomain can regulate histone acetyl transferase activity and interacts specifically with acetylated lysine residues. A key role for bromodomain proteins in maintaining normal proliferation is indicated by the implication of several bromodomain proteins in cancer, with four of these identified at translocation breakpoints. EST databases were searched for novel bromodomain genes. The sequence from one EST was used to initiate generation of a full-length clone from a testis cDNA library. The completed sequence encodes a predicted protein of 2781 amino acids, which, in addition to the bromodomain, harbors further motifs characteristic of a transcriptional coactivator: two PHD fingers and an extensive glutamine-rich acidic domain. There are several other regions that are conserved with the Caenorhabditis elegans putative protein F26H11, which may be functionally homologous. The novel gene, called BPTF, is expressed in all tissues examined as a 10.5-kb transcript. The protein has extensive identity with the smaller FAC1 protein, suggesting that the two either are derived from the same locus or are synonymous. BPTF has been mapped to 17q23. Functional domains found within BPTF are consistent with a role for this protein in hormonally regulated, chromatin-mediated regulation of transcription (Jones, 2000).

Transcription factors mediate their regulatory effects through interaction with DNA and numerous nuclear proteins. The fetal Alz-50 clone 1 (FAC1) protein, a novel DNA-binding protein with the capacity to repress transcription, is likely to function through a similar mechanism. Using the two-hybrid yeast screen, it has been shown that FAC1 interacts with the myc-associated zinc finger protein (ZF87/MAZ). This association was confirmed in vitro with recombinant protein. The ZF87/MAZ interaction domain was mapped to the region containing a putative nuclear localization signal (NLS) and nuclear export sequence (NES) of FAC1, using deletion mutants of the FAC1 protein. However, FAC1 recognizes a conformational interface that includes the proline/alanine-rich domain of ZF87/MAZ and the first zinc finger. Cotransfection of NIH3T3 cells with ZF87/MAZ and a luciferase reporter containing the SV40 promoter and enhancer results in an increase in transcriptional activation, suggesting ZF87/MAZ is able to recognize its consensus binding site present in the SV40 promoter. Cotransfection with FAC1 reduces the level of ZF87/MAZ-induced activation of the SV40 promoter in a dose dependent manner. A mutant FAC1, lacking the ZF87/MAZ interaction domain, does not alter ZF87/MAZ activation of the SV40 promoter. These data demonstrate that interaction between FAC1 and ZF87/MAZ alters the transactivation capacity of ZF87/MAZ. By immunoblot analysis, FAC1 and ZF87/MAZ exhibit similar tissue distribution and co-localize to pathologic structures in Alzheimer's disease brain. Coexpression of FAC1 and ZF87/MAZ suggest that interaction of these two proteins will have biological implications for gene regulation in neurodegeneration (Jordan-Sciutto, 2000).

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).

A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling

Lysine methylation of histones is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive chromatin domains. Trimethylation of histone H3 lysine 4 (H3K4me3) marks transcription start sites of virtually all active genes. The WD40-repeat protein WDR5 is important for global levels of H3K4me3 and control of HOX gene expression. A PHD finger of nucleosome remodelling factor (NURF), an ISWI-containing ATP-dependent chromatin-remodelling complex, mediates a direct preferential association with H3K4me3 tails. Depletion of H3K4me3 causes partial release of the NURF subunit, BPTF (bromodomain and PHD finger transcription factor), from chromatin and defective recruitment of the associated ATPase, SNF2L (also known as ISWI and SMARCA1), to the HOXC8 promoter. Loss of BPTF in Xenopus embryos mimics WDR5 loss-of-function phenotypes, and compromises spatial control of Hox gene expression. These results strongly suggest that WDR5 and NURF function in a common biological pathway in vivo, and that NURF-mediated ATP-dependent chromatin remodelling is directly coupled to H3K4 trimethylation to maintain Hox gene expression patterns during development. This study identifies a previously unknown function for the PHD finger as a highly specialized methyl-lysine-binding domain (Wysocka, 2005).

Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF

Mono-, di- and tri-methylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centered on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. This study demonstrates the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a PHD finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). Crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states is reported. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, key specificity-determining residues were identified from binding studies of H3(1-15)K4me3 with PHD finger point mutants. These findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins (Li, 2006).

Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells

This study has characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. Functional and physical links are provided between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. It is concluded that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo (Landry, 2008).

Because Bptf has been associated with the NURF complex, a known regulator of transcription, the data suggests that Bptf is required for the expression of gene targets in the developing visceral endoderm (VE) and distal visceral endoderm (DVE). In support of this hypothesis, it was shown that Bptf is required for the regulation of endogenous promoters and Smad responsive promoter elements in ES, P19 and MCF10CA1 cells in tissue culture. Smad-dependent gene targets include those essential for cell proliferation (p21) and those essential for DVE function (Cer1, Lefty1) and primitive streak (T, Fgf8, Gsc). Moreover, pulldown assays showed that components of the NURF complex have direct interactions with the Smad transcription factors and it is recruited to the promoters of Smad regulated genes under conditions of activation. Taken together, these data suggest that Bptf can directly regulate Smad regulated genes, likely through the functions of the NURF remodeling complex, via recruitment by the Smad transcription factors (It is also possible that other as yet unidentified Bptf-containing complexes distinct from NURF function in this pathway). To address the possibility that the effects on the Nodal/Smad pathway are indirect the expression of key components of the pathway was monitored in embryos. No significant changes were seen that could explain the observed defects in Smad signaling in the early embryo or during embryoid body differentiation in Bptf mutants. Consistent with these findings, the ISWI ATPase, a component of Drosophila NURF, ACF and CHRAC has been reported to be important for transmitting Dpp/TGFβ signals to stem cells in the Drosophila ovary. However, it is emphasized that the Bptf mutation likely affects many different pathways and biological processes, each of which may contribute to biological phenotypes. The challenge for the near future will be to uncover each of the many functions of Bptf in mammalian chromatin biology (Landry, 2008).

Four enzymes cooperate to displace histone H1 during the first minute of hormonal gene activation: NURF facilitates the progesterone receptor-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement

Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes, which is limited by the compaction of DNA in chromatin. Althought insight has been gained into how core histones and their modifications influence this process, the role of linker histones remains unclear. This study show that, within the first minute of progesterone action, a complex cooperation between different enzymes acting on chromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First, activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2 [activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 by the MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylase KDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, and kinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histone H2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) and expression arrays show that H1 displacement is required for hormone induction of most hormone target genes, some of which are involved in cell proliferation (Vicent, 2011).

These results contribute to a better comprehension of the molecular mechanisms of gene induction by describing the very initial steps of hormonal promoter activation. The data reveal an unexpected complexity in the interactions between enzymatic activities implicated in preparing the chromatin for full access of transcription factors. Apart from previously described enzymatic activities, at least four complexes act 1 min after hormone addition. An ATP-dependent chromatin remodeling complex (NURF), a protein kinase complex (Cdk2/CyclinA), a histone lysine demethylase (JARID1B/KDM5B), and a histone lysine methylase (MLL2 or MLL3)-containing complex cooperate in the displacement of histone H1 from the promoter, an important early step in gene induction by progestins (Vicent, 2011).

It has been shown, in T47D-MTVL cells treated with hormone for 5 min, PR interacts with an exposed HRE on the surface of a nucleosome positioned over the MMTV promoter and recruits Brg1/Brm-containing BAF complexes. This study demonstrates that NURF interacts with PR, and that recruitment of the NURF complex in the first minute following hormone addition is a requisite for subsequent binding of BAF and activation of mammary tumor virus (MMTV) and other progesterone target genes. NURF is anchored at the promoter of progesterone target genes by an interaction with H3K4me3, likely generated by the MLL2/3 histone lysine methylases of the ASCOM complex. This is reminiscent of the role of hormone-induced acetylation of H3K14 in anchoring the BAF complex. At both phases in activation of the promoter, a histone tail modification stabilizes the binding of an ATP-dependent chromatin remodeling complex to the target promoters (Vicent, 2011).

Another similarity between the two subsequent cycles of promoter chromatin remodeling relates to the role of protein kinases. It was found previously that hormone-induced activation of the Src/Ras/Erk cascade leads to phosphorylation of PR at S294 and activation of Msk1, which is targeted to the promoter by PR and phosphorylates H3 at S10, contributing to the displacement of a repressive complex containing HP1γ. This study shows that, prior to this event, 1 min after hormone, PR interacts with a complex of Cdk2 and CyclinA that phosphorylates PR at S400, is recruited to the promoter, and phosphorylates histone H1, leading to its displacement. Thus, there are two similar and consecutive cycles essential for transcriptional activation of hormone-dependent genes, both involving the collaboration between protein kinases, histone-modifying enzymes, and ATP-dependent chromatin remodelers. Each of the remodeling complexes is anchored at the promoter by different epigenetic marks: H3K4me3 established by MLL2/3 anchors NURF, and H3K14ac established by PCAF anchors BAF. The final output of the first cycle is to decompact the chromatin fiber by displacing histone H1, and the outcome of the second cycle is to open the nucleosome by displacing H2A/H2B dimers (Vicent, 2011).

The chromatin remodeling complex NURF has been shown to be necessary for both transcription activation and repression in vivo. Most reports on the role of NURF in gene regulation come from studies in Drosophila, where NURF is involved in the activation of several genes, including the homeotic selector gene engrailed, ultrabithorax, ecdysone-responsive genes, and the roX noncoding RNA. These studies were complemented with mechanistic studies using recombinant Drosophila NURF complex. In contrast, little is known regarding the mechanism of action of NURF in human cells, except for reports on a role in neuronal physiology. It was found that, in T47D-MTVL human breast cancer cells, NURF is essential for efficient hormone-dependent activation of several PR target genes, and is recruited to the target promoters via an interaction with PR. The BAF complex is also recruited to the MMTV promoter within minutes after progestin treatment, but the kinetics of loading of both chromatin remodelers are different. NURF is recruited after 1 min of hormone treatment, while BAF is loaded only after 5 min and its recruitment depends on NURF action. These findings highlight the notion of transcription initiation as a process involving consecutive cycles of enzymatic chromatin remodeling, where each enzyme complex is necessary at a given time point and catalyzes a particular remodeling step. These results support the existence in T47D-MTVL cells of several pools of PR, associated with the different chromatin remodelers. How the coordinated action of each PR population on target promoters is orchestrated is not well understood, but phosphorylation of the receptor by different kinases and post-translational modifications of nucleosomal histones could provide possible mechanisms (Vicent, 2011).

Although H3K4me3 marks transcription start sites (TSSs) of virtually all active genes the role of this modification during MMTV activation has been questioned. This study shows that, in T47D-MTVL cells, the MLL2/3-containing complex ASCOM is recruited to a target promoter after 1 min of hormone and increases H3K4me3. Experiments with siRNA knockdown, ChIP, and peptide pull-down assays showed that H3K4me3 is critical for NURF anchoring at the promoter. The very early and transient appearance of the H3K4me3 mark could explain the apparent controversy with previously published studies. It was found that the H3K4me3 signal observed at the MMTV promoter is due to the concerted recruitment of the ASCOM complex and the localized displacement of the H3K4me3/2/1 demethylase KDM5B. Knockdown of KDM5B increased the basal and hormone-dependent activity of PR target genes and caused an increase in H3K4me3 levels at the promoters in the absence of hormone (Vicent, 2011).

The molecular mechanism underlying hormone-induced displacement of KDM5B is unclear. It has been reported that KDM5B forms a complex with histone deacetylases (HDACs). Ir was shown previously that an HP1γ-containing complex is bound to the MMTV promoter prior to induction, and is displaced by phosphorylation of H3S10 catalyzed by hormone-activated Msk1. However, in coimmunoprecipitation experiments, no interaction between KDM5B and HP1γ was detected. Recently, it has been reported that PARP1 can parylate and inactivate KDM5B catalytic activity. Since nuclear receptors are known to activate PARP1, it is possible that this pathway participates in the inactivation and displacement of KDM5B following progestin treatment (Vicent, 2011).

The PHD finger present in the BPTF subunit of NURF acts as a highly specialized methyl lysine-binding domain critical for NURF loading. H3S10ph and H3K14ac, two other post-translational modifications present in the MMTV promoter chromatin after hormone addition, increase the binding of the PHD domain to H3K4me3. Binding of BPTF to acetylated lysines could be expected, as the protein contains a bromodomain in its C terminus, but the interaction with H3 phosphopeptides was not predicted, as BPTF does not encompass a consensus 14-3-3-like domain. Regarding the role of the H3K9me3 signal in NURF recruitment, peptide pull-down experiments showed no interaction of NURF components with the H3K9me3 mark. Moreover, either knockdown or inhibition of the methyltransferase G9a increased the basal level of transcription in several target genes without affecting the fold induction after hormone. The same effect was observed when cells were depleted of HP1γ, indicating that the H3K9me3 signal anchors HP1γ at the target chromatin (Vicent, 2011).

The NURF complex is recruited after 1 min of hormone, decreased after 2 min, and is almost undetectable after 5 min. How NURF is released from target chromatin is still unknown. Binding of NURF correlates closely with H3K4me3, and therefore a decrease in the trimethylation of H3K4 would explain NURF displacement. It has been proposed that methylation of histone H3R2 by PRMT6 and methylation of H3K4 by MLLs are mutually exclusive. Moreover, H3R2 methylation has been reported to block the binding of effectors that harbor methyl-specific binding domains, including PHD domains, chromodomains, and Tudor domains. Thus, the presence of the H3R2me2 mark could cooperate in erasing the H3K4me3 signal from the promoters and in competing for NURF binding, thus triggering NURF displacement (Vicent, 2011).

MMTV minichromosomes reconstituted with Drosophila embryo extracts were used previously to address the role of histone H1. Histone H1 increases nucleosome spacing and compacts the chromatin, hinders access of general transcription factors to the MMTV promoter, and thus inhibits basal transcription. In the presence of bound PR, H1 is phosphorylated by Cdk2 and subsequently is removed from the promoter upon transcription initiation. The kinase Cdk2 is known to phosphorylate histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions. Histone H1 phosphorylation by Cdk2 has been associated with hormone-dependent transcriptional activation. This study found that NURF facilitates the access of Cdk2/CyclinA to target promoter chromatin, and this could explain its role in H1 displacement from the MMTV promoter and from 15 other PR-binding sites that also contain NURF and recruit Cdk2 after hormone treatment. Along with the general effect of Cdk2 inhibition on gene regulation by progestins, these results support a very general role of Cdk2/CyclinA in histone H1 eviction during the initial steps of hormonal chromatin remodeling (Vicent, 2011).

There is evidence for a direct interaction between PR and Cdk2, CyclinA, or cyclinE that could explain how Cdk2/CyclinA is recruited to the target promoters. In the T47D-MTVL breast cancer cell line, a hormone-independent association of PR with Cdk2 was found and recruitment of CyclinA to this complex upon hormone addition. Therefore, PR could recruit Cdk2/CyclinA to the target promoter upon hormone addition. It is not known whether NURF and Cdk2/CyclinA form a single ternary complex with PR, or rather are in two different PR-associated complexes. Although by coimmunoprecipitation interaction of PR with both complexes was detected after 1 min of hormone addition, a more in-depth analysis performed at 1-min intervals at 30°C revealed that NURF is recruited before Cdk2/CyclinA. These results suggest that NURF recruitment is required for Cdk2/CyclinA loading at target promoters, and support the existence of two independent complexes (Vicent, 2011).

Although H1 displacement takes place locally, it could have a long-range effect on chromatin decompaction, as demonstrated with in vitro assembled condensed chromatin. Displacement of histone H1 could be a prerequisite for all subsequent steps in remodeling, as SWI/SNF remodeling has been reported to be inhibited by the presence of histone H1. A connection between ISWI-containing remodeling machineries and histone H1 dynamics has been reported previously in Drosophila. In this system, ISWI promotes the association of the linker histone H1 with chromatin. Along these lines, it is still possible that NURF is also involved in later steps during hormone induction by helping histone H1 deposition back at the promoter (Vicent, 2011).

How H1 binding is regulated and leads to a more open chromatin structure remains unclear. Some models proposed that Cdk2-dependent H1 phosphorylation leads to the decondensation of chromatin during interphase by disrupting the association of HP1γ with the chromatin fiber. A hormone-dependent displacement of HP1γ from the MMTV promoter was observed without changes in H3K9me3 levels. Whether H1 and Hp1γ are interacting as part of a common repressive complex requires further studies but constitutes an attracting hypothesis. In contrast, PARP-1 possesses the ability to disrupt chromatin structure by PARylating histones (e.g., H1 and H2B) and a variety of nuclear proteins involved in gene regulation. Both PARP-1 and H1 compete for binding to nucleosomes and exhibit a reciprocal pattern of binding at actively transcribed promoters: H1 is depleted and PARP-1 is enriched. Other post-translational modifications of H1 have been proposed to influence its binding and function. Histone H1 is acetylated at Lys 26 in vivo and can be deacetylated by the NAD+-dependent HDAC SirT1, promoting the formation of repressive heterochromatin. This effect was accompanied by an enrichment of H1 at the promoter, and the spreading of heterochromatin marks like H3K9me3 and H4K20me1 throughout the coding region (Vicent, 2011).

Regarding the NURF-mediated changes in chromatin structure, analysis of nucleosome profiles obtained by MNase digestion before hormone treatment showed a preferential location of nucleosomes overlapping with NURF and PR sites that is less pronounced after hormone activation, indicating that chromatin remodeling is involved (Vicent, 2011).

Analysis of the hormone-regulated genes that are affected by depletion of NURF reveals many genes involved in cell cycle and cell proliferation, which could mediate the proliferative response of breast cancer cells to progestins. This may explain the inhibition of cell proliferation in response to progestins that was observed in T47D cells depleted of NURF. A similar inhibition of the proliferative response has been observed in cells depleted of Cdk2. These results indicate that histone H1 displacement may be a prerequisite for the effects of progestins on cell proliferation, and therefore the enzymes involved in this process would be novel targets for the pharmacological control of breast cancer cell proliferation (Vicent, 2011).

A model of the current view of the initial steps in progesterone activation of the MMTV promoter is presented. Although the different steps of remodeling are depicted as a linear time sequence, it cannot be excluded that some of these process occur in parallel and in different time sequences in different target promoters. The model reflects the average time sequence in the cell population. Briefly, after hormone induction, activated PR carrying Erk and Msk1 binds first to the exposed HRE1 on the surface of the MMTV promoter nucleosome in a process that does not require chromatin remodeling. Along with the activated PR kinases, the NURF and ASCOM complexes are recruited to the promoter chromatin in one or several complexes. The combined action of ASCOM recruitment and KDM5B displacement enhances H3K4me3 and stabilizes NURF at the promoter. Other modifications, such as H3S10phos and H3K14ac produced by Msk1 and PCAF, could also contribute to NURF anchoring. Once at the promoter, NURF remodels the nucleosome and facilitates the access of PR and the associated Cdk2/CyclinA kinase, which phosphorylates histone H1 and promotes its displacement, contributing to unfolding of the chromatin fiber. Although it was observed that H3S10 phosphorylation by Msk1 plays a role in HP1γ displacement, it is possible that phosphorylation of histone H1 also contributes to this process. H1-depleted nucleosomes constitute a suitable substrate for recruitment of PR-BAF complexes and further remodeling events catalyzed by BAF and PCAF. H3K14 acetylation by PCAF promotes BAF anchoring. BAF mediates ATP-dependent displacement of histones H2A/H2B, and thus facilitates binding of NF1. Bound NF1 stabilizes the open conformation of the H3/H4 tetramer particle that exposes the previously hidden HREs, allowing synergistic binding of further PR-BAF-kinase complexes and PCAF (Vicent, 2011).

Finally, given that NURF is also recruited to the promoter 30 min after hormone addition, when no H1 is present, it cannot be excluded that NURF catalyzes later steps in chromatin remodeling involving histones or nonhistone chromatin proteins. Indeed, the current results indicate that NURF can act on MMTV minichromosomes lacking histone H1. In this respect, it remains to be established whether NURF and BAF fulfill partly redundant functions, cooperate on the same promoter, or, rather, are mutually exclusive (Vicent, 2011).

Chromatin remodeling complex NURF regulates thymocyte maturation

The maturation of T cells requires signaling from both cytokine and T-cell receptors to gene targets in chromatin, but how chromatin architecture influences this process is largely unknown. This study shows that thymocyte maturation post-positive selection is dependent on the nucleosome remodeling factor (NURF). Depletion of Bptf (bromodomain PHD finger transcription factor), the largest NURF subunit, in conditional mouse mutants results in developmental arrest beyond the CD4(+) CD8(int) stage without affecting cellular proliferation, cellular apoptosis, or coreceptor gene expression. In the Bptf mutant, specific subsets of genes important for thymocyte development show aberrant expression. Defects in DNase I-hypersensitive chromatin structures were also observed at Egr1, a prototypical Bptf-dependent gene that is required for efficient thymocyte development. Moreover, chromatin binding of the sequence-specific factor Srf (serum response factor) to Egr1 regulatory sites is dependent on Bptf function. Physical interactions between NURF and Srf suggest a model in which Srf recruits NURF to facilitate transcription factor binding at Bptf-dependent genes. These findings provide evidence for causal connections between NURF, transcription factor occupancy, and gene regulation during thymocyte development (Landry, 2011).

Enhancer of bithorax/NURF301: Biological Overview | Regulation | References

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