ATP-dependent Chromatin Remodeling Activities Research Articles

Gatad2b, associated with the neurodevelopmental syndrome GAND, plays a critical role in neurodevelopment and cortical patterning

GATAD2B (GATA zinc finger domain containing 2B) variants are associated with the neurodevelopmental syndrome GAND, characterized by intellectual disability (ID), infantile hypotonia, apraxia of speech, epilepsy, macrocephaly and distinct facial features. GATAD2B encodes for a subunit of the Nucleosome Remodeling and Histone Deacetylase (NuRD) complex. NuRD controls transcriptional programs critical for proper neurodevelopment by coupling histone deacetylase with ATP-dependent chromatin remodeling activity. To study mechanisms of pathogenesis for GAND, we characterized a mouse model harboring an inactivating mutation in Gatad2b. Homozygous Gatad2b mutants die perinatally, while haploinsufficient Gatad2b mice exhibit behavioral abnormalities resembling the clinical features of GAND patients. We also observed abnormal cortical patterning, and cellular proportions and cell-specific alterations in the developmental transcriptome in these mice. scRNAseq of embryonic cortex indicated misexpression of genes key for corticogenesis and associated with neurodevelopmental syndromes such as Bcl11b, Nfia and H3f3b and Sox5. These data suggest a crucial role for Gatad2b in brain development.

De novo variants in GATAD2A in individuals with a neurodevelopmental disorder: GATAD2A-related neurodevelopmental disorder.

GATA zinc finger domain containing 2A (GATAD2A) is a subunit of the nucleosome remodeling and deacetylase (NuRD) complex. NuRD is known to regulate gene expression during neural development and other processes. The NuRD complex modulates chromatin status through histone deacetylation and ATP-dependent chromatin remodeling activities. Several neurodevelopmental disorders (NDDs) have been previously linked to variants in other components of NuRD's chromatin remodeling subcomplex (NuRDopathies). We identified five individuals with features of an NDD that possessed de novo autosomal dominant variants in GATAD2A. Core features in affected individuals include global developmental delay, structural brain defects, and craniofacial dysmorphology. These GATAD2A variants are predicted to affect protein dosage and/or interactions with other NuRD chromatin remodeling subunits. We provide evidence that a GATAD2A missense variant disrupts interactions of GATAD2A with CHD3, CHD4, and CHD5. Our findings expand the list of NuRDopathies and provide evidence that GATAD2A variants are the genetic basis of a previously uncharacterized developmental disorder.

Relocalization of PU.1 Underlies the On- and Off-Tumor Effects of SWI/SNF Blockade in AML

SWI/SNF ATP-dependent chromatin remodeling activity has been identified as a strong dependency of acute myeloid leukemia (AML). In AML cell lines, genetic knockdown or chemical inhibition of the SWI/SNF ATPases SMARCA4 (BRG1) or SMARCA2 (BRM) by the selective small-molecule inhibitor BRM014 reduces proliferation and induces leukemic differentiation. These effects arise because SWI/SNF inhibition reduces DNA accessibility at binding sites of PU.1, a factor essential for driving high levels of MYC in leukemic cells. However, given that both SWI/SNF and PU.1 are essential in hematopoietic development, important questions regarding the use of SWI/SNF inhibitors against AML remain. First, the chromatin features that influence SWI/SNF-dependent PU.1 regulation within healthy hematologic and leukemic cells have not been established. Furthermore, the ability of SWI/SNF inhibitors to target leukemic cells within the bone marrow has remained uncertain, and the effects of these drugs in an immunocompetent setting have not been reported, despite the essential functions of SWI/SNF and PU.1 in normal hematopoiesis. Finally, the sensitivity of primary human AML samples and normal hematopoietic cells has also not been established. As a result, many questions regarding the nature of the therapeutic window for SWI/SNF inhibition against AML have remained unresolved. Here we evaluate SWI/SNF inhibition using BRM014 in clinically relevant models and examine the consequences of the deregulation of SWI/SNF-dependent PU.1 sites. We demonstrate that inhibition of SWI/SNF shifts PU.1 occupancy from enhancers to promoters in both leukemic and healthy hematopoietic cells. In AML, the shift of PU.1 away from enhancers to the promoters of differentiation-related genes results in loss of MYC expression and differentiation of leukemic cells. In non-leukemic mice, SWI/SNF inhibition suppresses PU.1-dependent B cell development and bone marrow-derived monocyte populations, reflecting off-tumor effects caused by SWI/SNF-dependent PU.1 relocalization. These effects influence peripheral immune cells, as well lineage-committed hematopoietic progenitors within the bone marrow. Moreover, we observe profound therapeutic response to SWI/SNF inhibition in an immunocompetent AML mouse model (Figure 1). In vivo, BRM014 induces differentiation of peripheral leukemic blasts and reduces the leukemic stem cell burden in bone marrow but also induces lymphocytopenia due to its off-tumor effects on PU.1. Nevertheless, the robust regression of leukemic burden seen in vivo over a short two-week treatment period provides a compelling justification for continued study of SWI/SNF inhibitors in the treatment of AML. Further, response of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and AML specimens highlight that normal hematopoiesis is affected at concentrations similar to those needed for therapeutic response in AML (Figure 2). Hence, hematopoietic and immune-related side effects, particularly at higher doses and over the duration of treatment, will be important considerations for clinical use of SWI/SNF inhibitors against AML and other tumors. Overall, our results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract ND14: Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers

Abstract Pharmacological profile and anti-tumor properties of FHD-286, a novel BAF inhibitor for the treatment of transcription factor-driven cancersMartin Hentemann, Rishi G. Vaswani, Lan Xu, Richard C. Centore, Luis M. M. Soares, Kana Ichikawa, Zhifang Li, Hong Fan, Jeremy Setser, David L. Lahr, Laura Zawadzke, Xueying Chen, Kimberly D. Barnash, Jordana Muwanguzi, Sarah Reilly, Neville Anthony, Gabriel J. Sandoval, Katharine Feldman, Ammar Adam, David Huang, Shawn Schiller, Kevin Wilson, Liyue Huang, Jessica Piel, Johannes Voigt, David S. Millan, Ho Man Chan, Ryan G. Kruger, Carl P. Decicco, Samuel Agresta, Steven F. BellonFoghorn Therapeutics, 500 Technology Square, Suite 700, Cambridge, MA 02139The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its ATP-dependent chromatin remodeling activity, BAF regulates the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. We have discovered and developed a compound that potently and selectively inhibits the ATPase components of the BAF complex, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). Mutational, structural, and biochemical studies demonstrated that this SMARCA4/SMARCA2 inhibitor acts through a unique allosteric mechanism. Pharmacologic inhibition of the BAF complex resulted in lineage-specific changes in chromatin accessibility in cancer cell lines from diverse origins. Phenotypic screening of cancer cell lines showed that uveal melanoma and hematological cancer cell lines were exquisitely sensitive to BAF inhibition. FHD-286 is orally bioavailable and demonstrates robust efficacy in multiple cell line and primary human tumor derived xenograft models at well-tolerated doses. In preclinical efficacy and toxicology studies, this first in class molecule demonstrates an acceptable therapeutic index which should allow for flexibility in clinical designs. FHD-286 is currently in two Phase I trials for both relapsed refractory AML and MDS, and metastatic uveal melanoma. Citation Format: Murphy Hentemann. Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND14.

Abstract 1224: Discovery of novel BAF inhibitors for the treatment of transcription factor-driven cancers

Abstract The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its ATP-dependent chromatin remodeling activity, BAF regulates the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. We have discovered and developed a novel series of compounds that potently and selectively inhibits the ATPase components of the BAF complex, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). Mutational, structural, and biochemical studies demonstrated that these SMARCA4/SMARCA2 inhibitors act through a unique allosteric mechanism. Pharmacologic inhibition of the BAF complex resulted in lineage-specific changes in chromatin accessibility in cancer cell lines from diverse origins. Phenotypic screening of cancer cell lines showed that uveal melanoma and hematological cancer cell lines were exquisitely sensitive to BAF inhibition. In the example of uveal melanoma, BAF inhibition resulted in the loss of accessibility at the binding sites of the SOX10 and MITF transcription factors, two essential proteins in supporting the proliferation and survival of uveal melanoma cells. Enhancer occupancy of SOX10 and MITF was reduced upon BAF inhibition, and subsequently, the melanocytic and pigmentation gene expression program regulated by these master transcription factors was suppressed. Finally, in a mouse xenograft model of uveal melanoma, BAF inhibition was well tolerated and resulted in dose-dependent tumor regression that correlated with pharmacodynamic modulation of BAF-target gene expression. These data provide the foundation for first-in-human studies of BAF ATPase inhibition as a novel therapeutic to treat uveal melanoma. Citation Format: Richard C. Centore, Luis M. Soares, Rishi G. Vaswani, Kana Ichikawa, Zhifang Li, Hong Fan, Jeremy Setser, David L. Lahr, Laura Zawadzke, Xueying Chen, Kimberly D. Barnash, Jordana Muwanguzi, Neville Anthony, Gabriel J. Sandoval, Katharine Feldman, Ammar Adam, David Huang, Shawn Schiller, Kevin Wilson, Johannes Voigt, Martin Hentemann, David S. Millan, Ho Man Chan, Steven F. Bellon, Carl P. Decicco, Lan Xu. Discovery of novel BAF inhibitors for the treatment of transcription factor-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1224.

Dot1 regulates nucleosome dynamics by its inherent histone chaperone activity in yeast

Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2–Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.

The structure of the core NuRD repression complex provides insights into its interaction with chromatin.

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin.

MTA family of proteins in DNA damage response: mechanistic insights and potential applications

The DNA damage, most notably DNA double-strand breaks, poses a serious threat to the stability of mammalian genome. Maintenance of genomic integrity is largely dependent on an efficient, accurate, and timely DNA damage response in the context of chromatin. Consequently, dysregulation of the DNA damage response machinery is fundamentally linked to the genomic instability and a likely predisposition to cancer. In turn, aberrant activation of DNA damage response pathways in human cancers enables tumor cells to survive DNA damages, thus, leading to the development of resistance of tumor cells to DNA damaging radio- and chemotherapies. A substantial body of experimental evidence has established that ATP-dependent chromatin remodeling and histone modifications play a central role in the DNA damage response. As a component of the nucleosome remodeling and histone deacetylase (NuRD) complex that couples both ATP-dependent chromatin remodeling and histone deacetylase activities, the metastasis-associated protein (MTA) family proteins have been recently shown to participate in the DNA damage response beyond its well-established roles in gene transcription. In this thematic review, we will focus on our current understandings of the role of the MTA family proteins in the DNA damage response and their potential implications in DNA damaging anticancer therapy.

Nucleosome remodeling by the SWI/SNF complex is enhanced by yeast High Mobility Group Box (HMGB) proteins

The regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key factors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues.

PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor

PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.

Williams syndrome is an epigenome-regulator disease

A human multi-protein complex (WINAC), composed of SWI/SNF components and DNA replication-related factors, that directly interacts with the vitamin D receptor (VDR) through the Williams syndrome transcription factor (WSTF), was identified with an ATP-dependent chromatin remodeling activity. This novel ATP-dependent chromatin remodeling complex facilitates VDR-mediated transrepression as well as transactivation with its ATP-dependent chromatin remodeling activity and promoter targeting property for the activator to access to the DNA. It also suggested that in this complex, WSTF serves as a signaling sensor to receive intra-cellular singalings to switch the activity of WINAC as well as WICH, another ATP-dependent chromatin remodeling complex containing hSNF2h. By making WSTF-deficient mice, some of the heart defects as well as abnormal calcium metabolism observed in Williams syndrome are attributed to the abnormal chromatin remodeling activity caused by WSTF deficiency. Thus, we would propose to designate Williams syndrome as an epigenome-regulator disease.

Chromatin Boundaries Require the Functional Cooperation Between hSET1 and NURF Complexes

AbstractAbstract 646Chromatin modification and remodeling activities play a central role in organizing nuclear functions in eukaryotic genome. Chromatin domains with characteristic epigenetic marks are organized by chromatin insulator. The chicken b-globin insulator, 5′HS4, is an excellent model system to study how insulator maintains gene function and prevents the encroachment of repressive heterochromatin. We showed previously that USF1/2 bound 5′HS4 insulator mediates chromatin barrier activity by recruiting and organizing active histone modifications in the chicken b-globin locus. However, it is not clear how insulator establishes such a barrier. To further understand the physical function of USF1, we purified the USF1-associated protein complexes. We found that USF1 forms a multiprotein complex with hSET1 and NURF exhibiting histone H3K4 methyltransferase and ATP-dependent chromatin remodeling activities, respectively. Both hSET1 and NURF complexes are specifically recruited to the 5′HS4 insulator by USF1 to retain the active chromatin structure. Knock-down of BPTF, a component of NURF complex, resulted in a rapid loss of barrier activity that protects a transgene from chromatin silencing. The loss of barrier by the BPTF knock-down is accompanied by an alternation of nucleosome positioning that expands the nucleosome to the nucleosome free linker region in the HS4 insulator site and increases repressive H3K9 dimethylation. Furthermore, the suppression of hSET1, a H3K4 methyltransferase, leads to not only a loss of barrier activity, but also a loss of H3K4 dimethylation and H3K9/K14 acetylation, as well as the recruitment of BPTF at chromatin insulator sites. Thus, our data reveal a molecular mechanism in which histone modifying enzyme hSET1 and chromatin remodeling complex NURF collaborate together to maintain the 5′HS4 chromatin barrier activity and to prevent the encroachment of adjacent heterochromatin. Disclosures:No relevant conflicts of interest to declare.

INO80 chromatin remodeling complex promotes the removal of UV lesions by the nucleotide excision repair pathway

The creation of accessible DNA in the context of chromatin is a key step in many DNA functions. To reveal how ATP-dependent chromatin remodeling activities impact DNA repair, we constructed mammalian genetic models for the INO80 chromatin remodeling complex and investigated the impact of loss of INO80 function on the repair of UV-induced photo lesions. We showed that deletion of two core components of the INO80 complex, INO80 and ARP5, significantly hampered cellular removal of UV-induced photo lesions but had no significant impact on the transcription of nucleotide excision repair (NER) factors. Loss of INO80 abolished the assembly of NER factors, suggesting that prior chromatin relaxation is important for the NER incision process. Ino80 and Arp5 are enriched to UV-damaged DNA in an NER-incision-independent fashion, suggesting that recruitment of the remodeling activity likely takes place during the initial stage of damage recognition. These results demonstrate a critical role of INO80 in creating DNA accessibility for the NER pathway and provide direct evidence that repair of UV lesions and perhaps most bulky adduct lesions requires chromatin reconfiguration.

DNA sequence encoded repression of rRNA gene transcription in chromatin

Eukaryotic genomes are packaged into nucleosomes that occlude DNA from interacting with most DNA-binding proteins. Nucleosome positioning and chromatin organization is critical for gene regulation. We have investigated the mechanism by which nucleosomes are positioned at the promoters of active and silent rRNA genes (rDNA). The reconstitution of nucleosomes on rDNA results in sequence-dependent nucleosome positioning at the rDNA promoter that mimics the chromatin structure of silent rRNA genes in vivo, suggesting that active mechanisms are required to reorganize chromatin structure upon gene activation. Nucleosomes are excluded from positions observed at active rRNA genes, resulting in transcriptional repression on chromatin. We suggest that the repressed state is the default chromatin organization of the rDNA and gene activation requires ATP-dependent chromatin remodelling activities that move the promoter-bound nucleosome about 22-bp upstream. We suggest that nucleosome remodelling precedes promoter-dependent transcriptional activation as specific inhibition of ATP-dependent chromatin remodelling suppresses the initiation of RNA Polymerase I transcription in vitro. Once initiated, RNA Polymerase I is capable of elongating through reconstituted chromatin without apparent displacement of the nucleosomes. The results reveal the functional cooperation of DNA sequence and chromatin remodelling complexes in nucleosome positioning and in establishing the epigenetic active or silent state of rRNA genes.

FACT and the Proteasome Promote Promoter Chromatin Disassembly and Transcriptional Initiation

The packaging of the eukaryotic genome into chromatin represses gene expression by blocking access of the general transcription machinery to the underlying DNA sequences. Accordingly, eukaryotes have developed a variety of mechanisms to disrupt, alter, or disassemble nucleosomes from promoter regions and open reading frames to allow transcription to occur. Although we know that chromatin disassembly from the yeast PHO5 promoter is triggered by the Pho4 activator, the mechanism is far from clear. Here we show that the Pho4 activator can occupy its nucleosome-bound DNA binding site within the PHO5 promoter. In contrast to the role of Saccharomyces cerevisiae FACT (facilitates chromatin transcription) complex in assembling chromatin within open reading frames, we find that FACT is involved in the disassembly of histones H2A/H2B from the PHO5 promoter during transcriptional induction. We have also discovered that the proteasome is required for efficient chromatin disassembly and transcriptional induction from the PHO5 promoter. Mutants of the degradation function of the proteasome have a defect in recruitment of the Pho4 activator, whereas mutants of the ATPase cap of the proteasome do recruit Pho4 but are still delayed for chromatin assembly. Finally, we rule out the possibility that the proteasome or ATPase cap is driving chromatin disassembly via a potential ATP-dependent chromatin remodeling activity.

HMGN Proteins Act in Opposition to ATP-Dependent Chromatin Remodeling Factors to Restrict Nucleosome Mobility

The high-mobility group N (HMGN) proteins are abundant nonhistone chromosomal proteins that bind specifically to nucleosomes at two high-affinity sites. Here we report that purified recombinant human HMGN1 (HMG14) and HMGN2 (HMG17) potently repress ATP-dependent chromatin remodeling by four different molecular motor proteins. In contrast, mutant HMGN proteins with double Ser-to-Glu mutations in their nucleosome-binding domains are unable to inhibit chromatin remodeling. The HMGN-mediated repression of chromatin remodeling is reversible and dynamic. With the ACF chromatin remodeling factor, HMGN2 does not directly inhibit the ATPase activity but rather appears to reduce the affinity of the factor to chromatin. These findings suggest that HMGN proteins serve as a counterbalance to the action of the many ATP-dependent chromatin remodeling activities in the nucleus.

CHD proteins: a diverse family with strong tiesThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

Chromodomain/helicase/DNA-binding domain (CHD) proteins have been identified in a variety of organisms. Despite common features, such as their chromodomain and helicase domain, they have been described as having multiple roles and interacting partners. However, a common theme for the main role of CHD proteins appears to be linked to their ATP-dependent chromatin-remodeling activity. Their actual activity as either repressor or activator, and their cell or gene specificity, is connected to their interacting partner(s). In this minireview, we attempt to match the members of the CHD family with the presence of structural domains, cofactors, and cellular roles in the regulation of gene expression, recombination, genome organization, and chromatin structure, as well as their potential activity in RNA processing.

Additional Sex Comb-like 1 (ASXL1), in Cooperation with SRC-1, Acts as a Ligand-dependent Coactivator for Retinoic Acid Receptor

Additional sex comb-like 1 (ASXL1, 170 kDa), a mammalian homolog of Drosophila ASX, was identified as a protein that interacts with retinoic acid receptor (RAR) in the presence of retinoic acid (RA). Systematic binding assays showed that the C-terminal nuclear receptor box (LVMQLL) of ASXL1 and the activation function-2 activation domain (AF-2 AD) core of the RAR are critical for ligand-dependent interaction. The interaction was confirmed using in vitro glutathione S-transferase pulldown and in vivo immunoprecipitation (IP) assays. Confocal microscopy revealed that ASXL1 localizes in the nucleus. In addition to the intrinsic transactivation function of ASXL1, its cotransfection together with an RA-responsive luciferase reporter increased the RAR activity. This ASXL1 activity appears to be mediated through the functional cooperation with SRC-1, as shown by GST pulldown, IP, chromatin IP, and transcription assays. In the presence of ASXL1, more acetylated histone H3 was accumulated on the RA-responsive promoter in response to RA. Finally, stable expression of ASXL1 increased the expression of endogenous RA-regulated genes and enhanced the antiproliferative potential of RA. Overall, these results suggest that ASXL1 is a novel coactivator of RAR that cooperates with SRC-1 and implicates it as a potential antitumor target of RA in RA-resistant cancer cells.

Analysis of Nucleosome Repositioning by Yeast ISWI and Chd1 Chromatin Remodeling Complexes

ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin remodeling activities in eukaryotes from yeast to man. Many of these complexes have been found to reposition nucleosomes but with different directionalities. We find that the yeast Isw1a, Isw2, and Chd1 enzymes preferentially move nucleosomes toward more central locations on short DNA fragments whereas Isw1b does not. Importantly, the inherent positioning properties of the DNA play an important role in determining where nucleosomes are relocated to by all of these enzymes. However, a key difference is that the Isw1a, Isw2, and Chd1 enzymes are unable to move nucleosomes to positions closer than 15 bp from a DNA end, whereas Isw1b can. We also find that there is a correlation between the inability of enzymes to move nucleosomes close to DNA ends and the preferential binding to nucleosomes bearing linker DNA. These observations suggest that the accessibility of linker DNA together with the positioning properties of the underlying DNA play important roles in determining the outcome of remodeling by these enzymes.

Nucleolar dominance: a model for rRNA gene silencing

Research over the last decade has demonstrated that the regulated packaging of DNA into chromatin is fundamental to keeping genes in an active/open or a more condensed/inactive conformation. Nucleosomes, the basic repeating unit of chromatin, contain two molecules each of canonical histones, H2A, H2B, H3, and H4. These are organized as an H3–H4 tetramer associated with two H2A–H2B dimers. The NH2 termini of histones protrude from the nucleosome, and are the target for a wide array of covalent modifications including acetylation, methylation, and phosphorylation. These modifications are applied and removed in a highly specific manner to generate what has been described as the histone code (Jenuwein and Allis 2001). This code is, in turn, read by chromatin-associated factors. For example, individual acetylated lysine residues are recognized by factors with bromodomains and methylated lysines by chromodomain-containing proteins. Additionally, the action of ATP-dependent chromatin remodeling activities can displace nucleosomes altering the accessibility of DNA within chromatin templates (Flaus and OwenHughes 2001). While most of this work has focused on protein-encoding genes transcribed by RNA polymerase II, more recent studies, including one by Earley et al. (2006) in this issue of Genes & Development, are aimed at elucidating the role that chromatin plays in determining the activity status of the genes that encode ribosomal RNAs (rRNAs). Eukaryotic genomes contain many rRNA gene copies, ranging from hundreds to thousands in some plants, organized in tandem arrays. rRNA genes are transcribed by RNA polymerase I (Pol I) into a precursor RNA (prerRNA) that encodes the three largest RNA components of ribosomes. Pre-rRNA coding sequences are separated by intergenic spacers (IGS) that can range in size from 3 kb in yeast to 30 kb in mammals. Pre-rRNA synthesis is regulated by elements including promoters, transcriptional enhancers, and terminators that are located within the IGS (Grummt 1999). Transcription of rRNA gene arrays results in formation of a nucleolus; consequently, they are termed nucleolar organizer regions (NORs). Importantly, not all NORs are transcriptionally active. Species with multiple NORs can vary the active proportion. Pre-rRNA is matured into 18S, 5.8S, and 28S rRNA by a precisely ordered series of events that includes both cleavages and targeted base modifications. These are required to ensure correct folding of rRNAs for ribosome assembly and function. These multiple steps in ribosome biogenesis require a bewildering array of proteins and small nucleolar RNAs that converge on transcriptionally active NORs, forming such a high concentration of ribonucleoprotein complexes that nucleoli are the most prominent feature of the eukaryotic nucleus. The electron micrographs of Miller and Beatty (1969) demonstrated that the coding regions of active repeats are fully loaded with Pol I. rRNA gene transcription accounts for ∼50% of nascent RNA synthesis in a cell. This high density of transcription and the recent demonstration that it is tightly coupled to pre-rRNA processing (Granneman and Baserga 2004) argues that rRNA gene chromatin needs to be especially accessible. As cells approach metaphase, Pol I transcription is actively shut down and the nucleolus disappears. It is at this point that the specialized nature of active rRNA gene chromatin is most dramatically revealed by the appearance of NORs as secondary constrictions on metaphase chromosomes (McClintock 1934).