A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation.

The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2 (myocyte enhancer factor 2), MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although it is known to reflect the action of chromatin modifiers. Here, we identify KAP1 (KRAB [Krüppel-like associated box]-associated protein 1)/TRIM28 (tripartite motif protein 28) as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only coactivators such as p300 and LSD1 but also corepressors such as G9a and HDAC1 (histone deacetylase 1), with promoter silencing as the net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the corepressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis.

Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation.

Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.

Cyclin-dependent kinase 1 activity coordinates the chromatin associated state of Oct4 during cell cycle in embryonic stem cells.

Cyclin-dependent kinase 1 (Cdk1) is indispensable for embryonic stem cell (ESC) maintenance and embryo development. Even though some reports have described a connection between Cdk1 and Oct4, there is no evidence that Cdk1 activity is directly linked to the ESC pluripotency transcription program. We recently reported that Aurkb/PP1-mediated Oct4 resetting is important to cell cycle maintenance and pluripotency in mouse ESCs (mESCs). In this study, we show that Cdk1 is an upstream regulator of the Oct4 phosphorylation state during cell cycle progression, and it coordinates the chromatin associated state of Oct4 for pluripotency-related gene expression within the cell cycle. Upon entry into mitosis, Aurkb in the chromosome passenger complex becomes fully activated and PP1 activity is inhibited downstream of Cdk1 activation, leading to sustaining Oct4(S229) phosphorylation and dissociation of Oct4 from chromatin during the mitotic phase. Cdk1 inhibition at the mitotic phase abnormally results in Oct4 dephosphorylation, chromosome decondensation and chromatin association of Oct4, even in replicated chromosome. Our study results suggest a molecular mechanism by which Cdk1 directly links the cell cycle to the pluripotency transcription program in mESCs.

Long non-coding RNA ChRO1 facilitates ATRX/DAXX-dependent H3.3 deposition for transcription-associated heterochromatin reorganization.

Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.

Abundance of C-terminal binding protein isoform is a prerequisite for exit from pluripotency in mouse embryonic stem cells.

Unlike lower organisms, mammals have 2 C-terminal binding protein (Ctbp) isoforms, Ctbp1 and Ctbp2. Ctbp2 is revealed as a key factor involved in determining cell fate decisions by regulating the epigenetic state in active embryonic stem cell (ESC) genes. However, the molecular mechanism underlying how Ctbp1 and Ctbp2 have different roles remains elusive. Here we demonstrate that Ctbp isoform abundance is important for mouse embryonic ESCs (mESCs) to exit from pluripotency. Temporal expression patterns of Ctbp isoforms were quite different; Ctbp2 is more highly expressed in mESCs and decreases during differentiation, while Ctbp1 is constantly expressed at a lower level. Ctbp2 knockdown, but not Ctbp1 knockdown, in mESCs resulted in impaired exit from pluripotency. Interestingly, Ctbp1 and Ctbp2 overexpression in Ctbp2-knockdown mESCs leads to exiting from pluripotency in a manner similar to that of wild-type mESCs. Quantification of Ctbp1 and Ctbp2 revealed that differentiation ability correlates with abundance of Ctbp isoform in undifferentiated mESCs, suggesting that a sufficient amount of Ctbp isoform is a prerequisite for exiting from pluripotency. The results support the contention that 2 redundant Ctbp isoforms regulate elaborate differentiation via temporally distinctive regulatory patterns in mESCs.-Suh, M. Y., Kim, T. W., Lee, H.-T., Shin, J., Kim, J.-H., Jang, H., Kim, H. J., Kim, S.-T., Cho, E.-J., Youn, H.-D. Abundance of C-terminal binding protein isoform is a prerequisite for exit from pluripotency in mouse embryonic stem cells.

OCT4 directly regulates stemness and extracellular matrix-related genes in human germ cell tumours.

Germ cell tumours (GCTs) are one of the most threatening malignancies in young men and women. Although several reports have suggested the importance of OCT4 in human GCTs, its role has not been clearly investigated on a molecular level. In this study, we revealed GCT-specific direct transcriptional target genes of OCT4. Conditional knockdown of OCT4 in GCT cell lines reduced cell proliferation by affecting both cell cycle and death. Knockdown of OCT4 also reduced stemness of GCTs, as assessed by the expression of other stemness factors, alkaline phosphatase staining, and tumour sphere formation ability. Analysis of whole mRNA expression patterns among GCT cells harbouring endogenous, depleted, and rescued OCT4 revealed 1133 OCT4 target genes in GCT. Combined analysis of both the chromatin binding signature of OCT4 and the genes whose expression levels were changed by OCT4 revealed 258 direct target genes of OCT4 in GCTs. In a similar way, 594 direct target genes in normal embryonic stem cells (ESCs) were identified. Among these two sets of OCT4 direct target genes, 38 genes were common between GCTs and ESCs, most of which were related to regulation of pluripotency, and 220 genes were specific to GCTs, most of which were related to focal adhesion and extracellular matrix organisation. These results provide a molecular basis for how OCT4 regulates GCT stemness and will aid our understanding of the role of OCT4 in other cancers.

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination.

Core Pluripotency Factors Directly Regulate Metabolism in Embryonic Stem Cell to Maintain Pluripotency.

Pluripotent stem cells (PSCs) have distinct metabolic properties that support their metabolic and energetic needs and affect their stemness. In particular, high glycolysis is critical for the generation and maintenance of PSCs. However, it is unknown how PSCs maintain and acquire this metabolic signature. In this study, we found that core pluripotency factors regulate glycolysis directly by controlling the expression of glycolytic enzymes. Specifically, Oct4 directly governs Hk2 and Pkm2, which are important glycolytic enzymes that determine the rate of glycolytic flux. The overexpression of Hk2 and Pkm2 sustains high levels of glycolysis during embryonic stem cell (ESC) differentiation. Moreover, the maintenance of high glycolysis levels by Hk2 and Pkm2 overexpression hampers differentiation and preserves the pluripotency of ESCs in the absence of leukemia inhibitory factor. Overall, our study identifies a direct molecular connection between core pluripotency factors and ESC metabolic signatures and demonstrates the significance of metabolism in cell fate determination.

Ctbp2 Modulates NuRD-Mediated Deacetylation of H3K27 and Facilitates PRC2-Mediated H3K27me3 in Active Embryonic Stem Cell Genes During Exit from Pluripotency.

For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. However, how this choreographed epigenetic remodeling at active embryonic stem cell (ESC) genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Furthermore, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment.

Modulation of lysine methylation in myocyte enhancer factor 2 during skeletal muscle cell differentiation.

Myocyte enhancer factor 2 (MEF2) is a family of transcription factors that regulates many processes, including muscle differentiation. Due to its many target genes, MEF2D requires tight regulation of transcription activity over time and by location. Epigenetic modifiers have been suggested to regulate MEF2-dependent transcription via modifications to histones and MEF2. However, the modulation of MEF2 activity by lysine methylation, an important posttranslational modification that alters the activities of transcription factors, has not been studied. We report the reversible lysine methylation of MEF2D by G9a and LSD1 as a regulatory mechanism of MEF2D activity and skeletal muscle differentiation. G9a methylates lysine-267 of MEF2D and represses its transcriptional activity, but LSD1 counteracts it. This residue is highly conserved between MEF2 members in mammals. During myogenic differentiation of C2C12 mouse skeletal muscle cells, the methylation of MEF2D by G9a decreased, on which MEF2D-dependent myogenic genes were upregulated. We have also identified lysine-267 as a methylation/demethylation site and demonstrate that the lysine methylation state of MEF2D regulates its transcriptional activity and skeletal muscle cell differentiation.

Phosphorylation and ubiquitination-dependent degradation of CABIN1 releases p53 for transactivation upon genotoxic stress.

CABIN1 acts as a negative regulator of p53 by keeping p53 in an inactive state on chromatin. Genotoxic stress causes rapid dissociation of CABIN1 and activation of p53. However, its molecular mechanism is still unknown. Here, we reveal the phosphorylation- and ubiquitination-dependent degradation of CABIN1 upon DNA damage, releasing p53 for transcriptional activation. The DNA-damage-signaling kinases, ATM and CHK2, phosphorylate CABIN1 and increase the degradation of CABIN1 protein. Knockdown or overexpression of these kinases influences the stability of CABIN1 protein showing that their activity is critical for degradation of CABIN1. Additionally, CABIN1 was found to undergo ubiquitin-dependent proteasomal degradation mediated by the CRL4DDB2 ubiquitin ligase complex. Both phosphorylation and ubiquitination of CABIN1 appear to be relevant for controlling the level of CABIN1 protein upon genotoxic stress.

Dissecting the roles of the histone chaperones reveals the evolutionary conserved mechanism of transcription-coupled deposition of H3.3.

The mammalian genome encodes multiple variants of histone H3 including H3.1/H3.2 and H3.3. In contrast to H3.1/H3.2, H3.3 is enriched in the actively transcribed euchromatin and the telomeric heterochromatins. However, the mechanism for H3.3 to incorporate into the different domains of chromatin is not known. Here, taking the advantage of well-defined transcription analysis system of yeast, we attempted to understand the molecular mechanism of selective deposition of human H3.3 into actively transcribed genes. We show that there are systemic H3 substrate-selection mechanisms operating even in yeasts, which encode a single type of H3. Yeast HIR complex mediated H3-specific recognition specificity for deposition of H3.3 in the transcribed genes. A critical component of this process was the H3 A-IG code composed of amino acids 87, 89 and 90. The preference toward H3.3 was completely lost when HIR subunits were absent and partially suppressed by human HIRA. Asf1 allows the influx of H3, regardless of H3 type. We propose that H3.3 is introduced into the active euchromatin by targeting the recycling pathway that is mediated by HIRA (or HIR), and this H3-selection mechanism is highly conserved through the evolution. These results also uncover an unexpected role of RI chaperones in evolution of variant H3s.

The role of tumor suppressor menin in IL-6 regulation in mouse islet tumor cells.

Menin is a gene product of multiple endocrine neoplasia type1 (Men1), an inherited familial cancer syndrome characterized by tumors of endocrine tissues. To gain insight about how menin performs an endocrine cell-specific tumor suppressor function, we investigated the possibility that menin was integrated in a cancer-associated inflammatory pathway in a cell type-specific manner. Here, we showed that the expression of IL-6, a proinflammatory cytokine, was specifically elevated in mouse islet tumor cells upon depletion of menin and Men(-/-) MEF cells, but not in hepatocellular carcinoma cells. Histone H3 lysine (K) 9 methylation, but not H3 K27 or K4 methylation, was involved in menin-dependent IL-6 regulation. Menin occupied the IL-6 promoter and recruited SUV39H1 to induce H3 K9 methylation. Our findings provide a molecular insight that menin-dependent induction of H3 K9 methylation in the cancer-associated interleukin gene might be linked to preventing endocrine-specific tumorigenesis.

Myogenic transcriptional activation of MyoD mediated by replication-independent histone deposition.

In mammals, the canonical histone H3 and the variant H3.3 are assembled into chromatin through replication-coupled and replication-independent (RI) histone deposition pathways, respectively, to play distinct roles in chromatin function. H3.3 is largely associated with transcriptionally active regions via the activity of RI histone chaperone, HIRA. However, the precise role of the RI pathway and HIRA in active transcription and the mechanisms by which H3.3 affects gene activity are not known. In this study, we show that HIRA is an essential factor for muscle development by establishing MyoD activation in myotubes. HIRA and Asf1a, but not CHD1 or Asf1b, mediate H3.3 incorporation in the promoter and the critical upstream regulatory regions of the MyoD gene. HIRA and H3.3 are required for epigenetic transition into the more permissive chromatin structure for polymerase II recruitment to the promoter, regardless of transcription-associated covalent modification of histones. Our results suggest distinct epigenetic management of the master regulator with RI pathway components for cellular differentiation.

p53 regulates glucose metabolism by miR-34a.

Cancer cells rely mainly on glycolysis rather than mitochondrial respiration for energy production, which is called the Warburg effect. p53 mutations are observed in about half of cancer cases, and p53 controls the cell cycle and cell death in response to cellular stressors. p53 has been emphasized as a metabolic regulator involved in glucose, glutamine, and purine metabolism. Here, we demonstrated metabolic changes in cancer that occurred through p53. We found that p53-inducible microRNA-34a (miR-34a) repressed glycolytic enzymes (hexokinase 1, hexokinase 2, glucose-6-phosphate isomerase), and pyruvate dehydrogenase kinase 1. Treatment with an anti-miR-34a inhibitor relieved the decreased expression in these enzymes following DNA damage. miR-34a-mediated inhibition of these enzymes resulted in repressed glycolysis and enhanced mitochondrial respiration. The results suggest that p53 has a miR-34a-dependent integrated mechanism to regulate glucose metabolism.

AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity.

CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

Determination of HIF-1α degradation pathways via modulation of the propionyl mark.

The hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxic stress under physiological and pathological conditions. HIF-1α protein stability is tightly regulated by the ubiquitin-proteasome system (UPS) and autophagy in normoxia, hypoxia, and the tumor environment to mediate the hypoxic response. However, the mechanisms of how the UPS and autophagy interplay for HIF-1α proteostasis remain unclear. Here, we found a HIF-1α species propionylated at lysine (K) 709 by p300/CREB binding protein (CBP). HIF-1α stability and the choice of degradation pathway were affected by HIF-1α propionylation. K709-propionylation prevented HIF-1α from degradation through the UPS, while activated chaperon-mediated autophagy (CMA) induced the degradation of propionylated and nonpropionylated HIF-1α. CMA contributed to HIF-1α degradation in both normoxia and hypoxia. Furthermore, the pan-cancer analysis showed that CMA had a significant positive correlation with the hypoxic signatures, whereas SIRT1, responsible for K709-depropionylation correlated negatively with them. Altogether, our results revealed a novel mechanism of HIF-1α distribution into two different degradation pathways. [BMB Reports 2023; 56(4): 252-257].

C-terminal binding protein-mediated transcriptional repression is regulated by X-linked inhibitor of apoptosis protein.

Inhibitors of Apoptosis Proteins (IAPs) are known as the key negative regulators of apoptosis. To explore new functions of IAPs, we sought to identify proteins that interact with Diap1 in insect S2 cells. We found that Diap1 bound to Drosophila C-terminal binding protein (dCtBP), which is a transcriptional co-repressor. CtBP1 also interacted with X-linked inhibitor of apoptosis protein (XIAP) in human cells. CtBPs were ubiquitinated by IAPs and targeted for proteasome-mediated degradation. Finally, the expression of CtBP1 target genes was regulated by XIAP expression. This is the first report to demonstrate that XIAP specifically regulates CtBP1, suggesting that XIAP may play a role in regulating CtBP1-mediated transcriptional repression by regulating the level of CtBP1.

A p53-inducible microRNA-34a downregulates Ras signaling by targeting IMPDH.

p53 is a well-known transcription factor that controls cell cycle arrest and cell death in response to a wide range of stresses. Moreover, p53 regulates glucose metabolism and its mutation results in the metabolic switch to the Warburg effect found in cancer cells. Nucleotide biosynthesis is also critical for cell proliferation and the cell division cycle. Nonetheless, little is known about whether p53 regulates nucleotide biosynthesis. Here we demonstrated that p53-inducible microRNA-34a (miR-34a) repressed inosine 5'-monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo GTP biosynthesis. Treatment with anti-miR-34a inhibitor relieved the expression of IMPDH upon DNA damage. Ultimately, miR-34a-mediated inhibition of IMPDH resulted in repressed activation of the GTP-dependent Ras signaling pathway. In summary, we suggest that p53 has a novel function in regulating purine biosynthesis, aided by miR-34a-dependent IMPDH repression.

The role of histone chaperones in osteoblastic differentiation of C2C12 myoblasts.

Cellular differentiation is a process in which the cells gain a more specialized shape, metabolism, and function. These cellular changes are accompanied by dynamic changes in gene expression programs. In most cases, DNA methylation, histone modification, and variant histones drive the epigenetic transition that reprograms the gene expression. Histone chaperones, HIRA and Asf1a, have a role for cellular differentiation by deposition of one of variant histones, H3.3, during myogenesis of murine C2C12 cells. In this study, we accessed the roles of histone chaperones and histone H3.3 in osteoblastic conversion of C2C12 myoblasts and compared their roles with those for myogenic differentiation. The unbiased analysis of the expression pattern of histone chaperones and variant histones proposed their uncommon contribution to each pathway. HIRA and Asf1a decreased to ∼50% and further diminished during differentiation into osteoblasts, while they were maintained during differentiation into myotubes. HIRA, Asf1a, and H3.3 were indispensable for expression of cell type-specific genes during conversion into osteoblasts or myotubes. RNA interference analysis indicated that histone chaperones and H3.3 were required for early steps of osteoblastic differentiation. Our results suggest that histone chaperones and variant histones might be differentially required for the distinct phases of differentiation pathway.