Heterochromatin Protein 1alpha Research Articles

Epigenetic marks uniquely tune the material properties of HP1α condensates

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1α) with DNA contributes to the formation of condensed chromatin states. HP1α localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1α and dictating emergent functions of its condensates remains to be understood. Here, we leverage a reductionist system, composed of modified and unmodified histone H3 peptides, HP1α, and DNA, to examine the contribution of specific epigenetic marks to phase behavior of HP1α. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1α condensates, whereas peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1α phase separation. Using fluorescence microscopy and rheological approaches, we further demonstrate that H3K9me3 histone peptides modulate the dynamics and viscoelastic network properties of HP1α condensates in a concentration-dependent manner. Additionally, in cells exposed to uniaxial strain, we find there to be a decreased ratio of nuclear H3K9me3 to HP1α. These data suggest that HP1α-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells and in response to mechanostimulation.

Transcriptional Control of HP1a By the RNA Binding Proteins Rbmx/L1 Maintain Chromatin State in Myeloid Leukemia

Mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to contribute to leukemia development (Prieto and Kharas, CSH, 2020). Previously we have identified the RBP RBMX (RNA binding motif protein, X-linked) in our shRNA in vivo screen using murine MLL-AF9 driven leukemia (Prieto et al, ASH abstract 2018). Here we uncover the role of RBMX and its retrogene RBMXL1 in normal hematopoiesis and leukemogenesis in mouse and human systems. To investigate RBMX function in normal hematopoiesis, we created an Rbmx Mx1-Cre conditional knockout model to specifically delete Rbmx in the hematopoietic system. We gender stratified our studies since Rbmx is sex-linked. Deletion of Rbmx in both female and male primary mice as well as in non-competitive transplant animals did not affect hematopoiesis. Additionally, Rbmx knockout (KO) leads to a mild reduction in multipotent progenitors (MPP2 and MPP4) in female competitive transplanted mice although no defects in long-term hematopoiesis was observed in male competitive transplanted mice. These data suggest that Rbmx is dispensable for normal hematopoiesis. To identify the role of RBMX in leukemogenesis we knockout Rbmx in MLL-AF9 murine leukemia cells and found it significantly reduced colony formation in vitro and delayed leukemogenesis in vivo, indicating that Rbmx is required for leukemia maintenance. We observed, however, that MLL-AF9 transformed cells from Rbmx KO donor mice showed no delay in leukemia initiation versus cells from wildtype. We then determined that while Rbmx deletion is effective with complete depletion of mRNA, the retrogene RBMXL1 expression is maintained in Rbmx deficient cells, which may compensate for Rbmx deletion in leukemia initiation. Indeed, depletion of RBMXL1 by shRNAs (KD) in Rbmx deficient leukemia cells results in a drastic reduction in colony formation, increased colony myeloid differentiation, and induced apoptosis in cells deficient for both RBMXL1 and RBMX compared to those only depleted of RBMX. Correspondingly, RBMXL1 KD in Rbmx deficient leukemia cells resulted in further delayed leukemogenesis in vivo, indicating that RBMXL1 is functionally redundant to RBMX and both genes are required for leukemia development and maintenance. We next investigated the role of RBMX/L1 in normal human hematopoietic and leukemia cells. We found that RBMX/L1 expression were higher in AML cell lines (n=10/11) and primary AML patient samples (n=2/4) compared to healthy individuals. RBMX/L1 KD by shRNAs led to a dramatic decrease in cell proliferation, induction of apoptosis and myeloid differentiation in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX/L1 depletion significantly delayed leukemogenesis in vivo of AML cell lines (median survival of 51.5 days in control vs. median 'not reached' in shRNA1 and shRNA2), and of primary leukemia cells derived from an AML patient (median 50 days in control vs. "not reached" in shRNA2). We next depleted RBMX/L1 in human CD34+ stem and progenitor cells and observed reduced colony formation but no increase in apoptosis. Taken together, our data suggest that RBMX/L1 are differentially required in leukemia cells versus normal cells in both human and mouse systems. To uncover the mechanism of RBMX/L1 function, we performed complex chromosomal karyotyping analysis of RBMX/L1 depleted MOLM13 cells and revealed increased metaphases with breaks and gaps. In addition, ATAC-seq analysis showed profound genome-wide changes in chromatin accessibility and compaction upon RBMX/L1 depletion. Analyses of RNA-seq and transcriptome-wide RBMX/L1 binding targets (PAR-CLIP; Liu et al. 2017) revealed that RBMX/L1 directly binds to mRNAs and affects transcription of multiple loci including the heterochromatin protein 1 alpha (HP1a) mRNA. Using single molecule RNA FISH, we uncovered that RBMX/L1 controls the nascent transcription of the HP1a locus. Forced HP1a expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through its downstream target HP1a. This study describes a novel genetic compensation phenomenon in leukemia and illuminate a mechanism for RBPs directly promoting transcription. Our results suggest RBMX/L1 as well as HP1a as potential novel therapeutic targets in myeloid malignancies. Disclosures Steidl: Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stelexis Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Research Funding; Pieris Pharmaceuticals: Consultancy. Kharas:Accent Therapeutics: Consultancy; 28-7: Research Funding.

P63 Transcription Factor Regulates Nuclear Shape and Expression of Nuclear Envelope-Associated Genes in Epidermal Keratinocytes

The maintenance of a proper nuclear architecture and three-dimensional organization of the genes, enhancer elements, and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by a marked decrease in expression of several nuclear envelope-associated components (Lamin B1, Lamin A/C, Sun1, Nesprin-3, Plectin) compared with controls. Furthermore, chromatin immunoprecipitation-quantitative PCR assay showed enrichment of p63 on Sun1, Syne3, and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks trimethylation on lysine 27 of histone H3, trimethylation on lysine 9 of histone H3, and heterochromatin protein 1-alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription toward the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture, and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression program in the epidermis.

Spatiotemporal regulation of Heterochromatin Protein 1-alpha oligomerization and dynamics in live cells.

Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the heterochromatin state. As consequence of playing a structural role in heterochromatin, HP1 proteins can have both an activating as well as repressive function in gene expression. Here we probe how oligomerisation of the HP1-α isoform modulates interaction with chromatin, by spatially resolved fluorescence correlation spectroscopy (FCS). We find from fluctuation analysis of HP1-α dynamics that this isoform exists as a dimer around the periphery of heterochromatin foci and these foci locally rotate with characteristic turn rates that range from 5–100ms. From inhibition of HP1-α homo-oligomerization we find the slow turn rates (20–100 ms) are dimer dependent. From treatment with drugs that disrupt or promote chromatin compaction, we find that HP1-α dimers spatially redistribute to favor fast (5–10 ms) or slow (20–100 ms) turn rates. Collectively our results demonstrate HP1-α oligomerization is critical to the maintenance of heterochromatin and the tunable dynamics of this HP1 isoform.

Overexpression Of The Histone Demethylase JMJD1C In Polycythemia Vera Contributes To NF-E2 Overexpression Via Epigenetic Dysregulation and An Auto-Regulatory Loop

Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have recently shown that NF-E2 overexpression in a murine model causes a phenotype similar to MPN in humans. This includes thrombocytosis, leukocytosis, expansion of the stem- and progenitor cell compartments as well as spontaneous transformation to acute leukemia. However, both the downstream pathways by which this transcription factor exerts its effects as well as the mechanisms leading to its overexpression in MPN patients remain incompletely characterized.We show here that the histone demethylases JMJD1C and JMJD2C constitute novel NF-E2 target genes. Using chromatin immunoprecipitation (ChIP) we demonstrate NF-E2 binding to the JMJD1C and JMJD2C loci. Both JMJD1C and JMJD2C protein levels are statistically significantly elevated in patients with polycythemia vera (PV) compared to healthy controls.JMJD1C and 2C proteins mediate the demethylation of histone H3K9, converting H3K9me2 via H3K9me to unmethylated H3K9. Consistent with increased demethylase activity, we observed statistically significantly decreased levels of global H3K9me and H3K9me2 in PV patients compared to healthy controls.We subsequently investigated whether the NF-E2 gene is regulated by epigenetic mechanisms and whether these are altered in PV patients. Indeed, using ChIP we demonstrate presence of the repressive H3K9me2 mark at three sites within the NF-E2 locus in healthy control granulocytes, where NF-E2 is expressed at low levels. These repressive histone marks are significantly reduced or completely absent in PV patients, which overexpress NF-E2. At the same time, binding of the repressive heterochromatin protein 1-alpha (HP1a) to the NF-E2 gene, while present in healthy controls, is significantly reduced in PV patients. HP1a chromatin binding is dependent on the presence of the H3K9me2 modification. Decreased levels of H3K9me2 and decreased HP1a binding are therefore both consistent with elevated NF-E2 expression in PV.We hypothesized that increased levels of the H3K9me2 demethylase JMJD1C in PV patients are responsible for the loss of this histone mark in the NF-E2 gene. Consistent with this model, we demonstrate significantly increased binding of JMJD1C to the NF-E2 locus in purified primary PV granulocytes.Our data demonstrate that the NF-E2 gene is regulated by epigenetic mechanisms and that these histone modifications are perturbed in PV patients. Moreover, NF-E2 participates in an auto-regulatory loop, by directly regulating transcription of the histone demethylase JMJD1C. Increased levels of NF-E2 thus cause increased JMJD1C expression. The JMJD1C protein, in turn, demethylates H3K9me2 residues in the NF-E2 locus, thereby further augmenting NF-E2 expression. Besides sustaining this novel autoregulatory loop, increased JMJD1C activity may contribute to MPN pathology by altering gene expression at additional loci.We sought to determine whether elevated NF-E2 levels could be normalized by pharmacological intervention. Treatment with the nucleoside analogue Decitabine reduced NF-E2 expression, and restored both the H3K9me2 marks and HP1a binding to the NF-E2 locus.These data provide a molecular rational for pre-clinical investigation of the effects of histone demethylase inhibitors and Decitabine on MPN cell biology and, subsequently, for a phase I trial investigating a combination of Decitabine and histone demethylase inhibitors in MPN patients. Disclosures:No relevant conflicts of interest to declare.

Modification of Integration Site Preferences of an HIV-1-Based Vector by Expression of a Novel Synthetic Protein

HIV-1-based lentiviral vectors are a promising tool for gene therapy. However, integration of a lentiviral vector into host cell genes may lead to the development of cancer. Therefore, control of integration site selection is critical to the successful outcome of gene therapy approaches that use these vectors. The discovery that integration site selection by HIV-1 and HIV-1-based vectors is controlled by the LEDGF/p75 protein has presented new opportunities to control integration site selection. In this study, we tested the hypothesis that a fusion protein containing the C-terminal HIV integrase-binding portion of LEDGF/p75, and the N-terminal chromodomain of heterochromatin protein-1alpha (HP1alpha), can target HIV-1 vector DNA outside of genes. We show that this fusion protein, termed TIHPLE, associates with the heterochromatin hallmark trimethylated Lys-9 of histone H3 (H3K9me3). Transient overexpression of TIHPLE alters integration site selection by an HIV-1-based vector and decreases the number of integration events that occur in genes. This change in integration site selection was achieved without a reduction in overall integration efficiency. Furthermore, we show that TIHPLE increases integration in the vicinity of H3K9me3 and in repetitive DNA sequences. These data provide a novel approach to address the problem of the tendency of retroviral vectors to integrate at undesirable sites of the human genome.

SUV39h-independent association of HP1β with fibrillarin-positive nucleolar regions

Heterochromatin protein 1 (HP1), which binds to sites of histone H3 lysine 9 (H3K9) methylation, is primarily responsible for gene silencing and the formation of heterochromatin. We observed that HP1 beta is located in both the chromocenters and fibrillarin-positive nucleoli interiors. However, HP1 alpha and HP1 gamma occupied fibrillarin-positive compartments to a lesser extent, corresponding to the distinct levels of HP1 subtypes at the promoter of rDNA genes. Deficiency of histone methyltransferases SUV39h and/or inhibition of histone deacetylases (HDACi) decreased HP1 beta and H3K9 trimethylation at chromocenters, but not in fibrillarin-positive regions that co-localized with RNA polymerase I. Similarly, SUV39h- and HDACi-dependent nucleolar rearrangement and inhibition of rDNA transcription did not affect the association between HP1 beta and fibrillarin. Moreover, the presence of HP1 beta in nucleoli is likely connected with transcription of ribosomal genes and with the role of fibrillarin in nucleolar processes.

The Metastasis Efficiency Modifier Ribosomal RNA Processing 1 Homolog B (RRP1B) Is a Chromatin-associated Factor

There is accumulating evidence for a role of germ line variation in breast cancer metastasis. We have recently identified a novel metastasis susceptibility gene, Rrp1b (ribosomal RNA processing 1 homolog B). Overexpression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in breast cancer. Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicted the outcome in three of four publicly available breast carcinoma gene expression data sets. In addition, we provide insights into the mechanism of RRP1B. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, including histone H1X, poly(ADP-ribose) polymerase 1, TRIM28 (tripartite motif-containing 28), and CSDA (cold shock domain protein A). Co-immunofluorescence and co-immunoprecipitation confirmed these interactions and also interactions with heterochromatin protein-1alpha and acetyl-histone H4 lysine 5. Finally, we investigated the effects of ectopic expression of an RRP1B allelic variant previously associated with improved survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wild type RRP1B in HeLa cells, the variant RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure.

Physical and Functional Interaction between Heterochromatin Protein 1α and the RNA-binding Protein Heterogeneous Nuclear Ribonucleoprotein U

By combining biochemical purification and mass spectrometry, we identified proteins associated with human heterochromatin protein 1alpha (HP1alpha) both in the nucleoplasm and in chromatin. Some of these are RNA-binding proteins, and among them is the protein heterogeneous nuclear ribonucleoprotein U (hnRNP U)/SAF-A, which is linked to chromatin organization and transcriptional regulation. Here, we demonstrate that hnRNP U is a bona fide HP1alpha-interacting molecule. More importantly, hnRNP U depletion reduces HP1alpha-dependent gene silencing and disturbs HP1alpha subcellular localization. Thus, our data demonstrate that hnRNP U is involved in HP1alpha function, shedding new light on the mode of action of HP1alpha and on the function of hnRNP U.

JAK2 phosphorylates histone H3Y41 and excludes HP1α from chromatin

Activation of Janus kinase 2 (JAK2) by chromosomal translocations or point mutations is a frequent event in haematological malignancies. JAK2 is a non-receptor tyrosine kinase that regulates several cellular processes by inducing cytoplasmic signalling cascades. Here we show that human JAK2 is present in the nucleus of haematopoietic cells and directly phosphorylates Tyr 41 (Y41) on histone H3. Heterochromatin protein 1alpha (HP1alpha), but not HP1beta, specifically binds to this region of H3 through its chromo-shadow domain. Phosphorylation of H3Y41 by JAK2 prevents this binding. Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1alpha at the same site. Tauhese results identify a previously unrecognized nuclear role for JAK2 in the phosphorylation of H3Y41 and reveal a direct mechanistic link between two genes, jak2 and lmo2, involved in normal haematopoiesis and leukaemia.

Heterochromatin Protein 1 (HP1) Proteins Do Not Drive Pericentromeric Cohesin Enrichment in Human Cells

Sister chromatid cohesion mediated by cohesin is essential for accurate chromosome segregation. Classical studies suggest that heterochromatin promotes cohesion, but whether this happens through regulation of cohesin remains to be determined. Heterochromatin protein 1 (HP1) is a major component of heterochromatin. In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region. To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA. We have also studied the consequences of treating human cells with drugs that change the histone modification profile of heterochromatin and thereby affect HP1 localization. Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis. However, depletion of HP1gamma leads to defects in mitotic progression.

Hypomethylation of subtelomeric regions in ICF syndrome is associated with abnormally short telomeres and enhanced transcription from telomeric regions

Telomeres and adjacent subtelomeric regions are packaged as heterochromatin in many organisms. The heterochromatic features include DNA methylation, histones H3-Lys9 (Lysine 9) and H4-Lys20 (Lysine 20) methylation and heterochromatin protein1 alpha binding. Subtelomeric DNA is hypomethylated in human sperm and ova, and these regions are subjected to de novo methylation during development. In mice this activity is carried out by DNA methyltransferase 3b (Dnmt3b). Mutations in DNMT3B in humans lead to the autosomal-recessive ICF (immunodeficiency, centromeric region instability, facial anomalies) syndrome. Here we show that, in addition to several satellite and non-satellite repeats, the subtelomeric regions in lymphoblastoid and fibroblast cells of ICF patients are also hypomethylated to similar levels as in sperm. Furthermore, the telomeres are abnormally short in both the telomerase-positive and -negative cells, and many chromosome ends lack detectable telomere fluorescence in situ hybridization signals from either one or both sister-chromatids. In contrast to Dnmt3a/b(-/-) mouse embryonic stem cells, increased telomere sister-chromatid exchange was not observed in ICF cells. Hypomethylation of subtelomeric regions was associated in the ICF cells with advanced telomere replication timing and elevated levels of transcripts emanating from telomeric regions, known as TERRA (telomeric-repeat-containing RNA) or TelRNA. The current findings provide a mechanistic explanation for the abnormal telomeric phenotype observed in ICF syndrome and highlights the link between TERRA/TelRNA and structural telomeric integrity.

Monitoring dynamic protein interactions with photoquenching FRET

The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy. We used this new method, photoquenching FRET (PQ-FRET), to define the dynamic interactions of the heterochromatin protein-1 alpha (HP1alpha) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) in regions of centromeric heterochromatin in mouse pituitary cells. The advantage of the PQ-FRET assay is that it provides simultaneous measurement of a protein's mobility, its exchange within macromolecular complexes and its interactions with other proteins in the living cell without the need for corrections based on reference images acquired from control cells.

Phosphorylation of the Linker Histone H1 by CDK Regulates Its Binding to HP1α

Two key components of mammalian heterochromatin that play a structural role in higher order chromatin organization are the heterochromatin protein 1alpha (HP1alpha) and the linker histone H1. Here, we show that these proteins interact in vivo and in vitro through their hinge and C-terminal domains, respectively. The phosphorylation of H1 by CDK2, which is required for efficient cell cycle progression, disrupts this interaction. We propose that phosphorylation of H1 provides a signal for the disassembly of higher order chromatin structures during interphase, independent of histone H3-lysine 9 (H3-K9) methylation, by reducing the affinity of HP1alpha for heterochromatin.

Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions

Mammalian circadian rhythms are based on transcriptional and post-translational feedback loops. Essentially, the activity of the transcription factors BMAL1 (also known as MOP3) and CLOCK is rhythmically counterbalanced by Period (PER) and Cryptochrome (CRY) proteins to govern time of day-dependent gene expression. Here we show that circadian regulation of the mouse albumin D element-binding protein (Dbp) gene involves rhythmic binding of BMAL1 and CLOCK and marked daily chromatin transitions. Thus, the Dbp transcription cycle is paralleled by binding of BMAL1 and CLOCK to multiple extra- and intragenic E boxes, acetylation of Lys9 of histone H3, trimethylation of Lys4 of histone H3 and a reduction of histone density. In contrast, the antiphasic daily repression cycle is accompanied by dimethylation of Lys9 of histone H3, the binding of heterochromatin protein 1alpha and an increase in histone density. The rhythmic conversion of transcriptionally permissive chromatin to facultative heterochromatin relies on the presence of functional BMAL1-CLOCK binding sites.

Mediation of Epstein-Barr virus EBNA-LP transcriptional coactivation by Sp100.

The Epstein-Barr virus (EBV) EBNA-LP protein is important for EBV-mediated B-cell immortalization and is a potent gene-specific coactivator of the viral transcriptional activator, EBNA2. The mechanism(s) by which EBNA-LP functions as a coactivator remains an important question in the biology of EBV-induced B-cell immortalization. In this study, we found that EBNA-LP interacts with the promyelocytic leukemia nuclear body (PML NB)-associated protein Sp100 and displaces Sp100 and heterochromatin protein 1alpha (HP1alpha) from PML NBs. Interaction between EBNA-LP and Sp100 was mediated through conserved region 3 in EBNA-LP and the PML NB targeting domain in Sp100. Overexpression of Sp100 lacking the N-terminal PML NB targeting domain, but not a mutant form of Sp100 lacking the HP1alpha interaction domain, was sufficient to coactivate EBNA2 in a gene-specific manner independent of EBNA-LP. These findings suggest that Sp100 is a major mediator of EBNA-LP coactivation. These studies indicate that modulation of PML NB-associated proteins may be important for establishment of latent viral infections, and also identify a convenient model system to investigate the functions of Sp100.

Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance.

The initiation of DNA replication in S phase requires the prior assembly of an origin recognition complex (ORC)-dependent pre-replicative complex on chromatin during G1 phase of the cell division cycle. In human cells, the Orc2 subunit localized to the nucleus as expected, but it also localized to centrosomes throughout the entire cell cycle. Furthermore, Orc2 was tightly bound to heterochromatin and heterochromatin protein 1alpha (HP1alpha) and HP1beta in G1 and early S phase, but during late S, G2 and M phases tight chromatin association was restricted to centromeres. Depletion of Orc2 by siRNA caused multiple phenotypes. A population of cells showed an S-phase defect with little proliferating cell nuclear antigen (PCNA) on chromatin, although MCM proteins remained. Orc2 depletion also disrupted HP1 localization, but not histone-H3-lysine-9 methylation at prominent heterochromatic foci. Another subset of Orc2-depleted cells containing replicated DNA arrested with abnormally condensed chromosomes, failed chromosome congression and multiple centrosomes. These results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle.

Identification and Characterization of ZFP-57, a Novel Zinc Finger Transcription Factor in the Mammalian Peripheral Nervous System

To isolate new zinc finger genes expressed at early stages of peripheral nerve development, we have used PCR to amplify conserved zinc finger sequences. RNA from rat embryonic day 12 and 13 sciatic nerves, a stage when nerves contain Schwann cell precursors, was used to identify several genes not previously described in Schwann cells. One of them, zinc finger protein (ZFP)-57, proved to be the homologue of a mouse gene found in F9 teratocarcinoma cells. Its mRNA expression profile within embryonic and adult normal and transected peripheral nerves, and its distribution in the rest of the nervous system is described. High levels of expression are seen in embryonic nerves and spinal cord. These drop rapidly during the first few weeks after birth, a pattern mirrored in other parts of the nervous system. ZFP-57 localizes to the nucleus of Schwann and other cells. The sequence contains an N-terminal Krüppel-associated box (KRAB) domain and ZFP-57 constructs containing green fluorescent protein reveal that the protein colocalizes with heterochromatin protein 1alpha to centromeric heterochromatin in a characteristic speckled pattern in NIH3T3 cells. The KRAB domain is required for this localization, because constructs lacking it target the protein to the nucleus but not to the centromeric heterochromatin. When fused to a heterologous DNA binding domain, the KRAB domain of ZFP-57 represses transcription, and full-length ZFP-57 represses Schwann cell transcription from myelin basic protein and P(0) promoters in co-transfection assays. Zfp-57 mRNA is up-regulated in Schwann cells in response to leukemia inhibitory factor and fibroblast growth factor 2.

Cytogenetic and immuno-FISH analysis of the 4q subtelomeric region, which is associated with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the shortening of a copy-number polymorphic array of 3.3 kb repeats (D4Z4) at one allelic 4q35.2 region. How this contraction of a subtelomeric tandem array causes FSHD is unknown but indirect evidence suggests that a short array has a cis effect on a distant gene or genes. It was hypothesized that the length of the D4Z4 array determines whether or not the array and a large proximal region are heterochromatic and thereby controls gene expression in cis. To test this, we used fluorescence in situ hybridization probes with FSHD and control myoblasts to characterize the distal portion of 4q35.2 with respect to the following: intense staining with the chromatin dye 4',6-diamidino-2-phenylindole; association with constitutively heterochromatic foci; extent of binding of heterochromatin protein 1alpha; histone H3 methylation at lysine 9 and lysine 4; histone H4 lysine 8 acetylation; and replication timing within S-phase. Our results indicate that 4q35.2 does not resemble constitutive heterochromatin in FSHD or control myoblasts. Furthermore, in these analyses, the allelic 4q35.2 regions of FSHD myoblasts did not behave differently than those of control myoblasts. Other models for how D4Z4 array contraction causes long-distance regulation of gene expression in cis need to be tested.

Human Ku70 Interacts with Heterochromatin Protein 1α

Ku is involved in the metabolism of DNA ends, DNA repair, and the maintenance of telomeres. It consists of a heterodimer of 70- and 80-kDa subunits. Recently we have demonstrated that Ku70 interacted with TRF2, a mammalian telomere-binding protein. Using the same yeast two-hybrid screening system, we now show that Ku70 also interacts with heterochromatin protein 1alpha (HP1alpha), a protein known to be associated with telomeres as well as heterochromatin. HP1 is a suppressor of the position effect variegation in Drosophila and acts as a transcriptional suppressor in mammalian cells. The interaction with Ku70 in the two-hybrid system was confirmed by a glutathione S-transferase pull-down study using bacterial recombinant proteins in vitro. The interaction was also reproduced in vivo in HeLa cells, where endogenous Ku70 coimmunoprecipitated with HP1alpha. This interaction was more effective in acidic pH and weakened considerably as the pH of the reaction buffer was elevated up to 7.5. Ku80 did not interact with HP1alpha directly. The interaction domains of Ku70 and HP1alpha included the Leu-Ser repeat (amino acids 200-385) and the chromo shadow domain, respectively. Ku70 was largely colocalized with transfected HP1alpha but not with a C-terminal deletion mutant, HP1alpha(Delta)C. In contrast to HP1alpha, Ku70 did not repress transcriptional activity of the reporter gene when tethered to DNA after transfection to mammalian cells. The implication of this interaction is discussed.