Alexander R. Ball

Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed “phenotypic” FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4–specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)–treated cells. We found that SUV39H1–mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1γ and cohesin are co-recruited to D4Z4 in an H3K9me3–dependent and cell type–specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type–specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1γ/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.

Cohesin mediates chromatin interactions that regulate mammalian β-globin expression.

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5′ and 3′ boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.

HP1: Heterochromatin binding proteins working the genome

Heterochromatin Protein 1 (HP1) is a transcriptional repressor that directly binds to the methylated lysine 9 residue of histone H3 (H3K9me), which is a hallmark histone modification for transcriptionally silenced heterochromatin. Studies of homologs in different organisms have provided significant insight into the function of HP1 and the role of H3K9me. Initially discovered to be a major constituent of heterochromatin important for gene silencing, HP1 is now known to be a dynamic protein that also functions in transcriptional elongation, centromeric sister chromatid cohesion, telomere maintenance, and DNA repair. Furthermore, recent studies have begun to uncover functional differences between HP1 variants and their H3K9me-independent mode of action. As our understanding of HP1 expands, however, conflicting data has also been reported that requires further reconciliation. Here we focus on some of the recent findings and controversies concerning HP1 functions in mammalian cells in comparison to studies in other organisms.

Identification of a Chromosome-Targeting Domain in the Human Condensin Subunit CNAP1/hCAP-D2/Eg7

ABSTRACT CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.

Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions.

Distinct functions of human cohesin-SA1 and cohesin-SA2 in double-strand break repair.

ABSTRACT Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G2-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesins intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells.

Revisiting the role of heterochromatin protein 1 in DNA repair

Heterochromatin protein 1 (HP1) is a conserved factor critical for heterochromatin organization and gene silencing. It is recruited to chromatin by its direct interaction with H3K9me (methylated lysine 9 residue of histone H3), an epigenetic mark for silenced chromatin. Now, Luijsterburg et al. (Luijsterburg, M.S., C. Dinant, H. Lans, J. Stap, E. Wiernasz, S. Lagerwerf, D.O. Warmerdam, M. Lindh, M.C. Brink, J.W. Dobrucki, et al. 2009. J. Cell Biol. 185:577–586) reveal a new H3K9me-independent role for HP1 in the DNA damage response, which is distinct from the one recently reported by Ayoub et al. (Ayoub, N., A.D. Jeyasekharan, J.A. Bernal, and A.R. Venkitaraman. 2008. Nature. 453:682–686).

Genetic and Epigenetic Characteristics of FSHD-Associated 4q and 10q D4Z4 that are Distinct from Non-4q/10q D4Z4 Homologs

Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of FSHD cases are linked to a decreased copy number of D4Z4 macrosatellite repeats on chromosome 4q (FSHD1). Less than 5% of FSHD cases have no repeat contraction (FSHD2), most of which are associated with mutations of SMCHD1. FSHD is associated with the transcriptional derepression of DUX4 encoded within the D4Z4 repeat, and SMCHD1 contributes to its regulation. We previously found that the loss of heterochromatin mark (i.e., histone H3 lysine 9 tri‐methylation (H3K9me3)) at D4Z4 is a hallmark of both FSHD1 and FSHD2. However, whether this loss contributes to DUX4 expression was unknown. Furthermore, additional D4Z4 homologs exist on multiple chromosomes, but they are largely uncharacterized and their relationship to 4q/10q D4Z4 was undetermined. We found that the suppression of H3K9me3 results in displacement of SMCHD1 at D4Z4 and increases DUX4 expression in myoblasts. The DUX4 open reading frame (ORF) is disrupted in D4Z4 homologs and their heterochromatin is unchanged in FSHD. The results indicate the significance of D4Z4 heterochromatin in DUX4 gene regulation and reveal the genetic and epigenetic distinction between 4q/10q D4Z4 and the non‐4q/10q homologs, highlighting the special role of the 4q/10q D4Z4 chromatin and the DUX4 ORF in FSHD.

Condensin I Recruitment to Base Damage-Enriched DNA Lesions Is Modulated by PARP1

Condensin I is important for chromosome organization and segregation in mitosis. We previously showed that condensin I also interacts with PARP1 in response to DNA damage and plays a role in single-strand break repair. However, whether condensin I physically associates with DNA damage sites and how PARP1 may contribute to this process were unclear. We found that condensin I is preferentially recruited to DNA damage sites enriched for base damage. This process is dictated by PARP1 through its interaction with the chromosome-targeting domain of the hCAP-D2 subunit of condensin I.

Localization of human SMC1 protein at kinetochores.

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesin is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesin associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesin dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesin components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis.