A Dimeric Structural Scaffold for PRC2-PCL Targeting to CpG Island Chromatin.

Diverse accessory subunits are involved in the recruitment of polycomb repressive complex 2 (PRC2) to CpG island (CGI) chromatin. Here we report the crystal structure of a SUZ12-RBBP4 complex bound to fragments of the accessory subunits PHF19 and JARID2. Unexpectedly, this complex adopts a dimeric structural architecture, accounting for PRC2 self-association that has long been implicated. The intrinsic PRC2 dimer is formed via domain swapping involving RBBP4 and the unique C2 domain of SUZ12. MTF2 and PHF19 associate with PRC2 at around the dimer interface and stabilize the dimer. Conversely, AEBP2 binding results in a drastic movement of the C2 domain, disrupting the intrinsic PRC2 dimer. PRC2 dimerization enhances CGI DNA binding by PCLs in pairs in vitro, reminiscent of the widespread phenomenon of transcription factor dimerization in active transcription. Loss of PRC2 dimerization impairs histone H3K27 trimethylation (H3K27me3) on chromatin at developmental gene loci in mouse embryonic stem cells.

Unique Structural Platforms of Suz12 Dictate Distinct Classes of PRC2 for Chromatin Binding.

Developmentally regulated accessory subunits dictate PRC2 function. Here, we report the crystal structures of a 120 kDa heterotetrameric complex consisting of Suz12, Rbbp4, Jarid2, and Aebp2 fragments that is minimally active in nucleosome binding and of an inactive binary complex of Suz12 and Rbbp4. Suz12 contains two unique structural platforms that define distinct classes of PRC2 holo complexes for chromatin binding. Aebp2 and Phf19 compete for binding of a non-canonical C2 domain of Suz12; Jarid2 and EPOP occupy an overlapped Suz12 surface required for chromatin association of PRC2. Suz12 and Aebp2 progressively block histone H3K4 binding to Rbbp4, suggesting that Rbbp4 may not be directly involved in PRC2 inhibition by the active H3K4me3 histone mark. Nucleosome binding enabled by Jarid2 and Aebp2 is in part accounted for by the structures, which also reveal that disruption of the Jarid2-Suz12 interaction may underlie the disease mechanism of an oncogenic chromosomal translocation of Suz12.

CircAGO2 promotes colorectal cancer progression by inhibiting heat shock protein family B (small) member 8 via miR-1-3p/retinoblastoma binding protein 4 axis.

This paper was to uncover the mechanism of circular RNA Argonaute 2 (circAGO2) in colorectal cancer (CRC) progression. The expression of circAGO2 was detected in CRC cells and tissues, and the relationship between clinicopathological features of CRC and circAGO2 level was evaluated. The growth and invasion of CRC cells and subcutaneous xenograft of nude mice were measured to evaluate the effect of circAGO2 on CRC development. Bioinformatics databases were applied to analyze levels of retinoblastoma binding protein 4 (RBBP4) and heat shock protein family B 8 (HSPB8) in cancer tissues. The relevance of circAGO2 and RBBP4 expression and the relationship between RBBP4 and HSPB8 during histone acetylation were assessed. The targeting relationship between miR-1-3p and circAGO2 or RBBP4 was predicted and confirmed. The effects of miR-1-3p and RBBP4 on biological functions of CRC cells were also verified. CircAGO2 was upregulated in CRC. CircAGO2 promoted the growth and invasion of CRC cells. CircAGO2 competitively bound to miR-1-3p and regulated RBBP4 expression, thus inhibiting HSPB8 transcription by promoting histone deacetylation. Silencing circAGO2 enhanced miR-1-3p expression and reduced RBBP4 expression, while suppression of miR-1-3p downgraded levels of miR-1-3p, up-regulated RBBP4, and facilitated cell proliferation and invasion in the presence of silencing circAGO2. RBBP4 silencing decreased RBBP4 expression and reduced proliferation and invasion of cells where circAGO2 and miR-1-3p were silenced. CircAGO2 overexpression decoyed miR-1-3p to increase RBBP4 expression, which inhibited HSPB8 transcription via histone deacetylation in HSPB8 promoter region, promoting proliferation and invasion of CRC cells.

Rbbp4 loss disrupts neural progenitor cell cycle regulation independent of Rb and leads to Tp53 acetylation and apoptosis.

BACKGROUND: Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression. Previous work indicated that Rbbp4 cooperates with the Rb tumor suppressor to block cell cycle entry. Here, we use genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival. RESULTS: Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers. Transgenic rescue of rbbp4 mutant embryos shows Rbbp4 is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ-H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. rbbp4; rb1 mutants show an additive effect on the number of M phase cells. In rbbp4 mutants, Tp53 acetylation is detected; however, Rbbp4 overexpression did not rescue DNA damage-induced apoptosis. CONCLUSION: Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0 independent of Rb. Tp53-dependent apoptosis in the absence of Rbpb4 correlates with Tp53 acetylation. Together these results suggest that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival through the regulation of Tp53 acetylation.

Photoaffinity Labeling-Based Chemoproteomic Strategy Reveals RBBP4 as a Cellular Target of Protopanaxadiol against Colorectal Cancer Cells.

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.

The topology of chromatin-binding domains in the NuRD deacetylase complex.

Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.

DREF Genetically Counteracts Mi-2 and Caf1 to Regulate Adult Stem Cell Maintenance.

Active adult stem cells maintain a bipotential state with progeny able to either self-renew or initiate differentiation depending on extrinsic signals from the surrounding microenvironment. However, the intrinsic gene regulatory networks and chromatin states that allow adult stem cells to make these cell fate choices are not entirely understood. Here we show that the transcription factor DNA Replication-related Element Factor (DREF) regulates adult stem cell maintenance in the Drosophila male germline. A temperature-sensitive allele of DREF described in this study genetically separated a role for DREF in germline stem cell self-renewal from the general roles of DREF in cell proliferation. The DREF temperature-sensitive allele caused defects in germline stem cell self-renewal but allowed viability and division of germline stem cells as well as cell viability, growth and division of somatic cyst stem cells in the testes and cells in the Drosophila eye. Germline stem cells mutant for the temperature sensitive DREF allele exhibited lower activation of a TGF-beta reporter, and their progeny turned on expression of the differentiation factor Bam prematurely. Results of genetic interaction analyses revealed that Mi-2 and Caf1/p55, components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, genetically antagonize the role of DREF in germline stem cell maintenance. Taken together, these data suggest that DREF contributes to intrinsic components of the germline stem cell regulatory network that maintains competence to self-renew.

Inhibition of Notch signaling by the p105 and p180 subunits of Drosophila chromatin assembly factor 1 is required for follicle cell proliferation.

Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. Here, we report another side of CAF1, wherein the subunits CAF1-p105 and CAF1-p180 (also known as CAF1-105 and CAF1-180, respectively) inhibit expression of Notch target genes and show this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt, also known as Pebbled), leading to Cut downregulation and inhibition of follicle cell mitosis. Our studies further show Notch is functionally responsible for these phenotypes observed in both the CAF1-p105- and CAF1-p180-deficient follicle cells. Moreover, we reveal that CAF1-p105- and CAF1-p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative-feedback regulatory loop between Cut and Hnt underlying CAF1-p105 and CAF-p180 regulation, which is crucial for follicle cell differentiation. In conclusion, our studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner.

The replicative histone chaperone CAF1 is essential for the maintenance of identity and genome integrity in adult stem cells.

Chromatin packaging and modifications are important to define the identity of stem cells. How chromatin properties are retained over multiple cycles of stem cell replication, while generating differentiating progeny at the same time, remains a challenging question. The chromatin assembly factor CAF1 is a conserved histone chaperone, which assembles histones H3 and H4 onto newly synthesized DNA during replication and repair. Here, we have investigated the role of CAF1 in the maintenance of germline stem cells (GSCs) in Drosophila ovaries. We depleted P180, the large subunit of CAF1, in germ cells and found that it was required in GSCs to maintain their identity. In the absence of P180, GSCs still harbor stem cell properties but concomitantly express markers of differentiation. In addition, P180-depleted germ cells exhibit elevated levels of DNA damage and de-repression of the transposable I element. These DNA damages activate p53- and Chk2-dependent checkpoints pathways, leading to cell death and female sterility. Altogether, our work demonstrates that chromatin dynamics mediated by CAF1 play an important role in both the regulation of stem cell identity and genome integrity.

Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex.

Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide.

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable.

Loss of RBBP4 results in defective inner cell mass, severe apoptosis, hyperacetylated histones and preimplantation lethality in mice†.

Retinoblastoma-binding protein 4 (RBBP4) (also known as chromatin-remodeling factor RBAP48) is an evolutionarily conserved protein that has been involved in various biological processes. Although a variety of functions have been attributed to RBBP4 in vitro, mammalian RBBP4 has not been studied in vivo. Here we report that RBBP4 is essential during early mouse embryo development. Although Rbbp4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts cannot hatch from the zona or can hatch but then arrest without further development. We find that while there is no change in proliferation or levels of reactive oxygen species, both apoptosis and histone acetylation are significantly increased in mutant blastocysts. Analysis of lineage specification reveals that while the trophoblast is properly specified, both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification. In summary, these findings demonstrate the essential role of RBBP4 during early mammalian embryogenesis.

A network-based variable selection approach for identification of modules and biomarker genes associated with end-stage kidney disease.

AIMS: Intervention for end-stage kidney disease (ESKD), which is associated with adverse prognoses and major economic burdens, is challenging due to its complex pathogenesis. The study was performed to identify biomarker genes and molecular mechanisms for ESKD by bioinformatics approach. METHODS: Using the Gene Expression Omnibus dataset GSE37171, this study identified pathways and genomic biomarkers associated with ESKD via a multi-stage knowledge discovery process, including identification of modules of genes by weighted gene co-expression network analysis, discovery of important involved pathways by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, selection of differentially expressed genes by the empirical Bayes method, and screening biomarker genes by the least absolute shrinkage and selection operator (Lasso) logistic regression. The results were validated using GSE70528, an independent testing dataset. RESULTS: Three clinically important gene modules associated with ESKD, were identified by weighted gene co-expression network analysis. Within these modules, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed important biological pathways involved in ESKD, including transforming growth factor-ß and Wnt signalling, RNA-splicing, autophagy and chromatin and histone modification. Furthermore, Lasso logistic regression was conducted to identify five final genes, namely, CNOT8, MST4, PPP2CB, PCSK7 and RBBP4 that are differentially expressed and associated with ESKD. The accuracy of the final model in distinguishing the ESKD cases and controls was 96.8% and 91.7% in the training and validation datasets, respectively. CONCLUSION: Network-based variable selection approaches can identify biological pathways and biomarker genes associated with ESKD. The findings may inform more in-depth follow-up research and effective therapy.

Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4.

BACKGROUND: Circular RNAs (circRNAs) are a novel type of noncoding RNAs and play important roles in tumorigenesis, including gastric cancer (GC). However, the functions of most circRNAs remain poorly understood. In our study, we aimed to investigate the functions of a new circRNA circ-DONSON in GC progression. METHODS: The expression of circ-DONSON in gastric cancer tissues and adjacent normal tissues was analyzed by bioinformatics method, qRT-PCR, Northern blotting and in situ hybridization (ISH). The effects of circ-DONSON on GC cell proliferation, apoptosis, migration and invasion were measured by using CCK8, colony formation, EdU, immunofluorescence (IF), FACS and Transwell assays. qRT-PCR and Western blotting were utilized to validate how circ-DONSON regulates SOX4 expression. ChIP, DNA fluorescence in situ hybridization (DNA-FISH) and DNA accessibility assays were used to investigate how circ-DONSON regulates SOX4 transcription. The interaction between circ-DONSON and NURF complex was evaluated by mass spectrum, RNA immunoprecipitation (RIP), pulldown and EMSA assays. Xenograft mouse model was used to analyze the effect of circ-DONSON on GC growth in vivo. RESULTS: Elevated expression of circ-DONSON was observed in GC tissues and positively associated with advanced TNM stage and unfavorable prognosis. Silencing of circ-DONSON significantly suppressed the proliferation, migration and invasion of GC cells while promoting apoptosis. circ-DONSON was localized in the nucleus, recruited the NURF complex to SOX4 promoter and initiated its transcription. Silencing of the NURF complex subunit SNF2L, BPTF or RBBP4 similarly attenuated GC cell growth and increased apoptosis. circ-DONSON knockdown inhibited GC growth in vivo. CONCLUSION: circ-DONSON promotes GC progression through recruiting the NURF complex to initiate SOX4 expression.

Histone methylation by PRC2 is inhibited by active chromatin marks.

The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.

Structural and functional characterization of the RBBP4-ZNF827 interaction and its role in NuRD recruitment to telomeres.

The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD-ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.

Retinoblastoma binding protein 4 up-regulation is correlated with hepatic metastasis and poor prognosis in colon cancer patients.

BACKGROUND: Retinoblastoma binding protein 4 (RBBP4) plays an essential role in the development of multiple cancers. However, its relationship with prognosis in colon cancer and colon cancer hepatic metastasis has not been elucidated. The aim of this study was to explore the relationship between RBBP4 expression and prognosis of colon cancer patients and to evaluate RBBP4 as a new prognostic marker in these patients. METHODS: Eighty colon cancer patients underwent surgical resection of the colon were enrolled. Among them, forty colon cancer patients suffered with hepatic metastasis. The colon cancer tissues, para-colon cancer tissues, and hepatic metastatic cancer tissues were collected from the pathological department for further analysis. The expression of RBBP4 proteins was examined by immunohistochemistry and correlated with clinicopathological parameters. The Cancer Genome Atlas (TCGA) database was used to validate the expression and explore its relationship with clinical characteristics. RESULTS: RBBP4 was up-regulated in the colon cancer tissues compared with the para-colon cancer tissues. The analysis of TCGA database verified the upregulation of RBBP4 in the colon cancer tissues and RBBP4 overexpression was correlated with nerve invasion and poor outcomes of chemotherapy. Moreover, the positive rate of RBBP4 expression in 40 colon cancer patients with hepatic metastasis was higher in the hepatic metastatic cancer tissues (39/40, 97.5%) than in the colon cancer tissues (26/40, 65.0%). Our clinicopathological analysis showed that RBBP4 expression was significantly correlated with vascular invasion, hepatic metastasis, and lymph node involvement (all P < 0.05). Additionally, the survival analysis demonstrated that RBBP4 over-expression was correlated with poor prognosis. CONCLUSIONS: RBBP4 was upregulated in the colon cancer. RBBP4 may be a novel predictor for poor prognosis of colon cancer and colon cancer hepatic metastasis.

DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.

In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence.

CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence.

MicroRNAs (miRNAs) recruit the CCR4-NOT complex, which contains two deadenylases, CCR4 and CAF1, to promote shortening of the poly(A) tail. Although both CCR4 and CAF1 generally have a strong preference for poly(A) RNA substrates, it has been reported from yeast to humans that they can also remove non-A residues in vitro to various degrees. However, it remains unknown how CCR4 and CAF1 remove non-A sequences. Herein we show that Drosophila miRNAs can promote the removal of 3'-terminal non-A residues in an exonucleolytic manner, but only if an upstream poly(A) sequence exists. This non-A removing reaction is directly catalyzed by both CCR4 and CAF1 and depends on the balance between the length of the internal poly(A) sequence and that of the downstream non-A sequence. These results suggest that the CCR4-NOT complex has an intrinsic activity to remove the 3'-terminal non-A modifications downstream from the poly(A) tail.

A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster.

The incorporation of CENP-A into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to our understanding of CENP-A/Cid loading pathways in Drosophila. Here, we report on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT and/or Caf1/Rbap48. Notably, we identified a novel complex consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. We show that Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition, we demonstrate that CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, our results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones.