CRL4B Interacts And Coordinates With SIN3A/HDAC Complex To Drive Cell Cycle Progression

Histone modification, an important aspect of epigenetic regulation, plays important roles in a variety of biological processes. Histone modifications include methylation, acetylation, phosphyorylation, ubiquitination and sumoylation. Histone acetylation,occurring mostly at lysine residues on the amino-terminal tails of histone H3and H4, is linked to the opening of chromatin and activation of gene expression either by altering their affinity to DNA, or by creating binding sites for the proteins that regulate chromatin accessibility. The antagonistic activities of two types of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), control the reversible acetylation state. HATs catalyze the acetylation of histones and other proteins, while HDACs catalyze the removal of the acetyl moieties from acetylated proteins. To date,18mammalian HDAC isoforms have been characterized and are classified into class I, class II, class III and class IV. Among them, Class I HDACs include HDAC1, HDAC2, HDAC3and HDAC8. HDAC1and HDAC2have redundant functions, and have been found to be members of a number of deacetylase complexes, including the SIN3A/HDAC complex, the NuRD complex, and the CoREST complex. Cells lacking both HDAC1and HDAC2show G1cell cycle arrest that was accompanied by up- regulation of CDK inhibitors p21and p57.CRLs (CULLIN RING Ubiquitin Ligase) are the largest class of ubiquitin ligases involved in cell cycle progression, signal transduction, development and DNA damage repair. As the scaffold of the complex, cullins belong to a conserved family. Human cells express eight different cullins, including CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7and PARC. The N-terminal domain of CUL4binds the substrate adaptor DNA damage binding protein1(DDB1), which recruits various substrate-recognition protein(DCAF). CRL4targets different substrates for proteasomal degradation or for protein modification, and thus regulates a broad variety of physiologically and developmentally-controlled processes. Recent studies have illustrated that CRL4is an important epigenetic regulator, participating in DNA methylation, heterochromatin formation and histone methylation. Although earlier studies focused on the redundant function and common substrates of CUL4members, CUL4B has been recently reported to function distinctly from CUL4A in transcriptional repression because of its unique N-terminus. CUL4B ablation blocked the degradation of WDR5, a core subunit of histone H3lysine4(H3K4) methyltransferase complex, increased the enrichment of trimethylated H3K4(H3K4me3) on the neuronal gene promoters and induced their expression. Recently, we showed that CRL4B functions as a transcriptional co-repressor of tumor suppressors by monoubiquitylating H2AK119and coordinating with either PRC2complex or DNMT/HP1/SUV39H1to regulate histone methylation or DNA methylation to promote tumorigenesis.We here show that CUL4B depletion can inhibit cell proliferation by up-regulating the transcriptions of Cdknla and Cdknlc. Importantly, we identified the physical and functional coordination of CRL4B complex with SIN3A/HDAC to exert its repressive effect.Part OneGeneration of Cul4b-null embryonic fibroblasts and characterization of their phenotypesOur previous results showed that Cul4b null embryos died by E9.5. H&E staining of E7.5embryos confirmed that Cul4b null embryos are developmentally retarded. A recent report showed that silencing of Cul4b expression in an extra-embryonic cell line resulted in accumulation of p21protein. To further understand the critical roles of CUL4B in regulating cell cycle progression, mouse embryonic fibroblasts were generated and analyzed.1. We generated the viable Cul4b-null mice by crossing Cul4b floxed mice to Sox2-Cre transgenic mice, and prepared the wild type and Cul4b-nnll embryonic fibroblasts (MEFs) from littermate embryos of E13.5, which is illustrated as Cul4bf/Y and Cul4b-/Y, respectively. Inducible conditional knockouts were generated by crossing Cul4bflox/flox female to CAG-Cre/Esrl male mice. Genotypes were determined by PCR. qRT-PCR and Western blot analyses showed that Cul4b was efficiently knockout, while Cul4a was detected at comparable levels between wild-type and Cul4b-null MEFs.2. To determine whetherthe two types of MEFs have different proliferations, we seeded equal numbers of Cul4bf/Y and Cul4b-/Y MEFs into replicate plates and monitored the fractions of Cul4b-positive cells at different passages by immunofluorescence staining of CUL4B. We observed that the percentages of CUL4B-positive cells were significantly increased with the increased passage numbers, indicating that Cul4b-/Y MEFs were selected against. The proliferations of Cul4bf/Y and Cul4b-/Y MEFs were further evaluated by MTT and EdU cell proliferation assays. The proliferation of Cul4b-/Y MEFs was significantly reduced compared with the MEFs from wild-type littermates. Similar results were obtained with knockout MEFs generated with4-Hydroxytamoxifen(OHT)-inducible Cre recombinase. Cul4b-/Y MEFs showed an increased accumulation of cells in G1phase of cell cycle, with concomitant decreases in the proportion of those in G2and S phases compared with WT MEFs. Furthermore, we found that Cul4b null MEFs displayed aflattened, enlarged morphology and expressed SA-β-Gal, but no positive signal in Tunel assay.Taken together, these results indicate that CUL4B positively regulates the cell cycle progression and negatively regulates cell senescence.Part TwoCUL4B promotes cell cycle progression through transcriptionally repressing the expression of CDKN1A A recent report showed that silencing of Cul4b expression in an extra-embryonic cell line resulted in accumulation of p21protein. Thus, we examined the effect of CUL4B depletion on the expression of this CDK. inhibitor:1. Western bloting demonstrated that loss of CUL4B resulted in a significant accumulation of p21. Similar results were obtained with CUL4B-knockdown HEK293and HeLa cells. Importantly, quantitative RT-PCR (qRT-PCR) assay indicated that the transcript level of Cdknla, which encodes p21, was also up-regulated in Cul4b-null MEFs, and similar results were obtained in CUL4B-knockdown HEK293and HeLa cells, suggesting that CUL4B may repress the expression of CDKN1A at transcriptional level.2. Since CDKN1A is a major transcriptional target of p53, we next examined whether the elevation of CDKN1A was due to increased activation of p53in CUL4B-deficient cells. Western blotting showed that there was no difference in the level of p53in CUL4B-deficient cells or Cul4b-/Y MEFs as compared with controls. In addition, treatment with p53inhibitor (Pifithrin-a) did not block the up-regulation of p21in CUL4B-knockdown293cells, supporting the notion that the up-regulation of p21in CUL4B-deficient cells was independent of p53activation.3. To examine the effect of CUL4B on the degradation of p21protein, treatment with proteasome inhibitor MG132did not block the reduction of p21in CUL4B-overexpressed cells. Furthermore, we measured p21decay rates by adding cycloheximide to the culture medium to inhibit new protein synthesis. We failed to detect any significant stabilization p21.These results demonstrate that CUL4B mediates the expression of CDKNlA via transcriptional regulation instead of proteolysis, and that is independent of p53activation.4. To determine whether the compromised proliferation observed in CUL4B-deficient cells might result from the upregulation of p21, we performed rescue experiment by transfecting siRNA targeting Cdknla or control siRNA into Cul4b-/Y MEFs. We found that knockdown of Cdknla could partially restore the proliferation defects caused by Cul4b depletion as indicated by EdU assays, supporting that CUL4B promotes cell proliferation at least partially through repressing the transcription of Cdknla in MEFS.Part Three CUL4B complex is physically associated with SIN3A/HDAC complex The CDKNIA has been shown to be targeted by SIN3A/HDAC complex. Our previous results showed that RbAp46/48, which are components of SIN3A/HDAC complex, could interact with CRL4B complex. We thus testes whether CRL4B and SIN3A/HDAC complexes physically interact.1. Immunoprecipitation (IP) with antibodies against CUL4B or DDB1was followed by immnuoblotting (IB) with antibodies against key components of SIN3A/HDAC complex,including HDAC1, HDAC2, SIN3A, SAP180, SAP130, SAP45, SAP30and RBP1. The results showed that CUL4B and DDB1could be co-immunoprecipitated with the examined components in the SIN3A/HDAC complex. Reciprocally, IP with antibodies against HDAC1, HDAC2, SIN3A, SAP130, SAP30, SAP45, SAP180or RBP1followed by immunoblotting with antibodies against CUL4B or DDB1also revealed that the proteins in the two complexes could be co-immunoprecipitated. These results suggest that CRL4B complex is physically associated with SIN3A/HDAC complex.2. To further investigate the molecular basis for the interaction between CRL4B and SIN3A/HDAC complexes, GST-pulldown assays were performed. With RbAp46/48proteins known to physically interact with SIN3A, serving as the positive control, these experiments revealed that both CUL4B and DDB1could directly interact with SIN3A, but not with the other components tested.3. SIN3A interacts with a large number of transcriptional factors and coregulators. To detect which region of SIN3A protein can directly interact with CUL4B/DDB1, we generated three GST-fused SIN3A constructs that contains different domains, GST-SIN3A S1(1-400amino acids), S2(401-657amino acids) and S3(658-1269amino acids). GST-pulldown experiments demonstrated that the fragment S2, which contains PAH3and HID domain of SIN3A, could directly bind to CUL4B and DDB1. Interestingly, the cullin domain of CUL4B was not required for its binding to SIN3A.4. The physical association between CRL4B and SIN3A/HDAC prompted us to determine the role of CRL4B in the histone deacetylation activity. To this end, Flag-CUL4B was expressed in HEK293cells and cellular extracts were prepared by affinity purification. As expected, the components of both CRL4B and SIN3A/HDAC complexes were detected in the affinity purified fractions.We then determined histone deacetylase activities of affinity-purified CUL4B-associated complex in vitro. The results showed that the amounts of acetylated H3(AcH3) and H4(AcH4), AcH4K8and AcH4K16were gradually decreased with increased amount of CUL4B-purified fractions, supporting the notion that CUL4B-associated complex has histone deacetylase activity. And this activity could be inhibited by TSA. We next examined the effect of CUL4B deletion on histone acetylation in MEFs. Compared with those in wild-type MEFs, increased acetylation levels of the bulk histone3and4were observed in Cul4b-null MEFs. We also examined the acetylation levels of individual residues and observed significantly increased levels of acetylation at H3K27, H4K8and H4K16in Cul4b-null MEFs, as compared with wild-type MEFs. The overall acetylation levels of H3and H4were similarly increased in CUL4B-knockdown HEK293and HeLa cells, although the increase in acetylation at the histone H4residues examined was more obvious.Collectively, these results demonstrate a physical association between CRL4B and SIN3A/HD AC with SIN3A serving as a linker to CUL4B and DDB1. And CRL4B may function as a transcriptional repressor by associating with this histone deacetylase complex.Part FourSIN3A/HDAC is involved in CUL4B-mediated transcriptional regulation of CDKNlAThe data above show that CRL4B complex is physically associated with SIN3A/HDAC complex, and that CUL4B depletion increases histone acetylation. We then examined whether SIN3A/HDAC is involved in CUL4B-mediated transcriptional regulation.1. We transfected Gal4-CUL4B into HeLa cells with stable expression of luciferase reporter (Gal4-UAS cells), and treated cells with or without TSA. While expression of Gal4-CUL4B led to a remarkable decrease in the expression of reporter gene in a dose dependent manner, TSA treatment could efficiently attenuate the reduction due to the expression of Gal4-CUL4B, suggesting that TSA-sensitive HDACs are involved in CUL4B-mediated transcriptional regulation. Furthermore, knockdown of HDAC1/2also led to a significant reduction in the transcriptional repressive activity of CUL4B. We then performed quantitative chromatin immuno-precipitation (qChIP) assay in Gal4-UAS cells and found that transfection of Gal4-CUL4B resulted in an increase in the recruitment of DDB1, HDAC1, HDAC2and SIN3A. Consequently, significant increased H2AK119ub1and decreased levels of acetylated histones, including AcH3, AcH4, AcH4K8and AcH4K16, were detected when Gal4-CUL4B was expressed. Consistent with these observation, TS A treatment could partially block the reduction of CDKN1A expression caused by CUL4B overexpression at both protein and RN A levels. Knockdown of SIN3A could also cause derepression of the CDKN1A. Importantly, simultaneous knockdown of SIN3A and CUL4B resulted in more severe effect than individual knockdown. Taken together, these results suggest that CRL4B coordinates with SIN3A/HDAC1/2to repress the transcription of target genes.2. In order to define the functional interplay between CRL4B and SIN3A/HDAC complexes in the transcriptional repression of CDKN1A gene, we first performed qChIP assay to profile the binding pattern of CRL4B and SIN3A/HDAC on a region of13kb that surrounds the transcriptional start site of CDKN1A. The qChIP assay revealed that the occupancy sites of these proteins overlap and that the binding of CRL4B and SIN3A/HDAC on CDKNlA promoter peaked in the region around-800bp to-300bp, suggesting that CRL4B and SIN3A/HDAC complexes co-occupy the CDKN1A promoter in the same complex. ChIP/Re-ChIP assays further confirmed this notion. We next examined the effect of CUL4B depletion on the recruitment of SIN3A/HDAC complex to the CDKNlA promoter, and found that knockdown of CUL4B resulted in marked reduction in the recruitment of HDAC1, HDAC2and SIN3A to the CDKNlA promoter. Consistent with these observations, greatly decreased H2AK119ubl and increased levels of acetylated histones, including AcH3, AcH4, AcH4K8and AcH4K16, were detected at the CDKNlA promoter upon CUL4B knockdown. On the other hand, knockdown of SIN3A or HDAC1/2did not affect the recruitment of CUL4B and DDB1at CDKN1A promoter. Collectively, these data support the argument that CRL4B contributes to histone deacetylation probably by facilitating the retention of HDAC1/2at CDKN1A promoter.3. To further understand the functional association between CRL4B and SIN3A/HDAC complexes, we examined the expression of10cell cycle regulators, which are previously identified as HDAC1/2target genes in primary fibroblasts, in Cul4b-null MEFs. Among10genes examined,5were up-regulated in Cul4b-/Y MEFs and4-OHT+MEFs compared with controls, suggesting that CRL4B complex is involved in regulating a subset of HDAC1/2target genes. Interestingly, Cdknlc, another member of Cip/Kip CKI family, was also significantly up-regulated in Cul4b-null MEFs compared with wide types. The negative effect of CUL4B on expression of CDKNlC was confirmed in CUL4B-knockdown and CUL4B-overexpressing HEK293cells. ChIP/Re-ChIP assay showed that CRL4B and SIN3A/HDAC complexes co-occupied at CDKNlC promoter as well.In conclusion, our study revealed that CRL4B interacts with SIN3A and promotes the recruitment and stable retention of SIN3A/HDAC to the CDKNIAand CDKNlC promoters, leading to increased H2AK119monoubiquitination and decreased histone acetylation and consequently epigenetic silencing of CDK inhibitors, and thus driving cell cycle progression.