Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation.

The super elongation complex (SEC) containing P-TEFb plays a critical role in regulating transcription elongation. AFF1 and AFF4, members of the AF4/FMR2 family, act as central scaffold proteins of SEC and are associated with various human diseases. However, their precise roles in transcriptional control remain unclear. We here reveal differences in the genomic distribution patterns of AFF1 and AFF4 around transcription start sites (TSSs). AFF1 mainly binds upstream of the TSSs, while AFF4 is enriched downstream of the TSSs. Notably, disruption of AFF4 results in slow elongation and early termination in a subset of AFF4 bound active genes, whereas AFF1 deletion leads to fast elongation and transcriptional readthrough in the same gene subset. Additionally, AFF1 knockdown increases AFF4 levels at chromatin, and vice versa. In summary, these findings demonstrate that AFF1 and AFF4 function antagonistically to regulate Pol II transcription.

Young LINE-1 transposon 5' UTRs marked by elongation factor ELL3 function as enhancers to regulate naïve pluripotency in embryonic stem cells.

LINE-1s are the major clade of retrotransposons with autonomous retrotransposition activity. Despite the potential genotoxicity, LINE-1s are highly activated in early embryos. Here we show that a subset of young LINE-1s, L1Md_Ts, are marked by the RNA polymerase II elongation factor ELL3, and function as enhancers in mouse embryonic stem cells. ELL3 depletion dislodges the DNA hydroxymethylase TET1 and the co-repressor SIN3A from L1Md_Ts, but increases the enrichment of the Bromodomain protein BRD4, leading to loss of 5hmC, gain of H3K27ac, and upregulation of the L1Md_T nearby genes. Specifically, ELL3 occupies and represses the L1Md_T-based enhancer located within Akt3, which encodes a key regulator of AKT pathway. ELL3 is required for proper ERK activation and efficient shutdown of naïve pluripotency through inhibiting Akt3 during naïve-primed transition. Our study reveals that the enhancer function of a subset of young LINE-1s controlled by ELL3 in transcription regulation and mouse early embryo development.

The super elongation complex (SEC) mediates phase transition of SPT5 during transcriptional pause release.

Release of promoter-proximally paused RNA Pol II into elongation is a tightly regulated and rate-limiting step in metazoan gene transcription. However, the biophysical mechanism underlying pause release remains unclear. Here, we demonstrate that the pausing and elongation regulator SPT5 undergoes phase transition during transcriptional pause release. SPT5 per se is prone to form clusters. The disordered domain in SPT5 is required for pause release and gene activation. During early elongation, the super elongation complex (SEC) induces SPT5 transition into elongation droplets. Depletion of SEC increases SPT5 pausing clusters. Furthermore, disease-associated SEC mutations impair phase properties of elongation droplets and transcription. Our study suggests that SEC-mediated SPT5 phase transition might be essential for pause release and early elongation and that aberrant phase properties could contribute to transcription abnormality in diseases.

QSER1 preserves the suppressive status of the pro-apoptotic genes to prevent apoptosis.

Activation of the pro-apoptotic genes by the p53 family is a critical step in induction of apoptosis. However, the molecular signaling underlying their suppression remains largely unknown. Here, we report a general role of QSER1 in preventing apoptosis. QSER1 is widely up-regulated in multiple cancers, and its up-regulation correlates with poor clinic outcomes. QSER1 knockdown significantly promotes apoptosis in both p53 wild type and mutant cancer cells. Interestingly, we show that QSER1 and p53 occupy distinct cis-regulatory regions in a common subset of the pro-apoptotic genes, and function antagonistically to maintain their proper expression. Furthermore, we identify a key regulatory DNA element named QSER1 binding site in PUMA (QBP). Deletion of QBP de-represses PUMA and induces apoptosis. Mechanistically, QSER1 functions together with SIN3A to suppress PUMA in a p53-dependent and -independent manner, suggesting that QSER1 inhibition might be a potential therapeutic strategy to induce apoptosis in cancers.

Phase Separation Properties in Transcriptional Organization.

Transcription is of great importance to stress response, fate control, and development, involving the functional cooperation of a large number of transcription factors and cofactors. Transcription machineries assemble rapidly to respond to the physiological and functional needs of cells. Recently, phase-separated biomolecular condensates have emerged as a universal biophysical basis for the spatiotemporal coordination of various cellular activities, including transcription. Here, we summarize and discuss recent advances in understanding of how phase separation contributes to RNA polymerase II (Pol II)-mediated transcriptional regulation, with a focus on the physical properties and dynamics of transcriptional condensates.

ZFP281-BRCA2 prevents R-loop accumulation during DNA replication.

R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to genome instability. Here, we show that ZFP281 cooperates with BRCA2 in preventing R-loop accumulation to facilitate DNA replication in embryonic stem cells. ZFP281 depletion reduces PCNA levels on chromatin and impairs DNA replication. Mechanistically, we demonstrate that ZFP281 can interact with BRCA2, and that BRCA2 is enriched at G/C-rich promoters and requires both ZFP281 and PRC2 for its proper recruitment to the bivalent chromatin at the genome-wide scale. Furthermore, depletion of ZFP281 or BRCA2 leads to accumulation of R-loops over the bivalent regions, and compromises activation of the developmental genes by retinoic acid during stem cell differentiation. In summary, our results reveal that ZFP281 recruits BRCA2 to the bivalent chromatin regions to ensure proper progression of DNA replication through preventing persistent R-loops.

Epigenetic reorganization during early embryonic lineage specification.

BACKGROUND: Dynamic chromatin reorganization occurs during two waves of cell lineage specification process, blastocyst formation and gastrulation, to generate distinct cell types. Epigenetic defects have been associated with severe developmental defects and diseases. How epigenetic remodeling coordinates the two lineage specification waves is becoming uncovered, benefiting from the development and application of new technologies including low-input or single-cell epigenome analysis approached in the past few years. OBJECTIVE: In this review, we aim to highlight the most recent findings on epigenetic remodeling in cell lineage specification during blastocyst formation and gastrulation. METHODS: First, we introduce how DNA methylation dynamically changes in blastocyst formation and gastrulation and its function in transcriptional regulation lineage-specific genes. Then, we discuss widespread remodeling of histone modification at promoters and enhancers in orchestrating the trajectory of cell lineage specification. Finally, we review dynamics of chromatin accessibility and 3D structure regulating developmental gene expression and associating with specific transcription factor binding events at stage specific manner. We also highlight the key questions that remain to be answered to fully understand chromatin regulation and reorganization in lineage specification. CONCLUSION: Here, we summarize the recent advances and discoveries on epigenetic reorganization and its roles in blastocyst formation and gastrulation, and how it cooperates with the lineage specification, painting from global sequencing data from mouse in vivo tissues.

Suppression of the NTS-CPS1 regulatory axis by AFF1 in lung adenocarcinoma cells.

Upregulation of the neuropeptide neurotensin (NTS) in a subgroup of lung cancers has been linked to poor prognosis. However, the regulatory pathway centered on NTS in lung cancer remains unclear. Here we identified the NTS-specific enhancer in lung adenocarcinoma cells. The AF4/FMR2 (AFF) family protein AFF1 occupies the NTS enhancer and inhibits NTS transcription. Clustering analysis of lung adenocarcinoma gene expression data demonstrated that NTS expression is highly positively correlated with the expression of the oncogenic factor CPS1. Detailed analyses demonstrated that the IL6 pathway antagonizes NTS in regulating CPS1. Thus, our analyses revealed a novel NTS-centered regulatory axis, consisting of AFF1 as a master transcription suppressor and IL6 as an antagonist in lung adenocarcinoma cells.

RNF219 interacts with CCR4-NOT in regulating stem cell differentiation.

Regulation of RNA stability plays a crucial role in gene expression control. Deadenylation is the initial rate-limiting step for the majority of RNA decay events. Here, we show that RING finger protein 219 (RNF219) interacts with the CCR4-NOT deadenylase complex. RNF219-CCR4-NOT exhibits deadenylation activity in vitro. RNA-seq analyses identify some of the 2-cell-specific genes and the neuronal genes significantly downregulated upon RNF219 knockdown, while upregulated after depletion of the CCR4-NOT subunit CNOT10 in mouse embryonic stem (ES) cells. RNF219 depletion leads to impaired neuronal lineage commitment during ES cell differentiation. Our study suggests that RNF219 is a novel interacting partner of CCR4-NOT and required for maintenance of ES cell pluripotency.

ENL initiates multivalent phase separation of the super elongation complex (SEC) in controlling rapid transcriptional activation.

Release of paused RNA polymerase II (Pol II) requires incorporation of the positive transcription elongation factor b (P-TEFb) into the super elongation complex (SEC), thus resulting in rapid yet synchronous transcriptional activation. However, the mechanism underlying dynamic transition of P-TEFb from inactive to active state remains unclear. Here, we found that the SEC components are able to compartmentalize and concentrate P-TEFb via liquid-liquid phase separation from the soluble inactive HEXIM1 containing the P-TEFb complex. Specifically, ENL or its intrinsically disordered region is sufficient to initiate the liquid droplet formation of SEC. AFF4 functions together with ENL in fluidizing SEC droplets. SEC droplets are fast and dynamically formed upon serum exposure and required for rapid transcriptional induction. We also found that the fusion of ENL with MLL can boost SEC phase separation. In summary, our results suggest a critical role of multivalent phase separation of SEC in controlling transcriptional pause release.

Rapid Delivery of Nanobodies/VHHs into Living Cells via Expressing In Vitro-Transcribed mRNA.

Intracellular antigen labeling and manipulation by antibodies have been long-thought goals in the field of cell research and therapy. However, a central limitation for this application is that antibodies are not able to penetrate into the cytosol of living cells. Taking advantages of small sizes and unique structures of the single-domain antibodies, here, we presented a novel approach to rapidly deliver the nanobody/variable domain of heavy chain of heavy-chain antibody (VHH) into living cells via introducing its coding mRNA, which was generated by in vitro transcription. We demonstrated that actin-green fluorescent proteins (GFP) and Golgi-GFP can be recognized by the anti-GFP nanobody/VHH, vimentin can be recognized by the anti-vimentin nanobody/VHH, and histone deacetylase 6 (HDAC6) can be recognized by the anti-HDAC6 nanobody/VHH, respectively. We found that the anti-GFP nanobody expressed via in vitro-transcribed (IVT) mRNA can be detected in 3 h and degraded in 48 h after transfection, whereas the nanobody expressed via plasmid DNA, was not detected until 24 h after transfection. As a result, it is effective in delivering the nanobody through expressing the nanobody/VHH in living cells from its coding mRNA.

ZFP281 Recruits MYC to Active Promoters in Regulating Transcriptional Initiation and Elongation.

The roles of the MYC transcription factor in transcriptional regulation have been studied intensively. However, the general mechanism underlying the recruitment of MYC to chromatin is less clear. Here, we found that the Krüppel-like transcription factor ZFP281 plays important roles in recruiting MYC to active promoters in mouse embryonic stem cells. At the genome scale, ZFP281 is broadly associated with MYC, and the depletion of ZFP281 significantly reduces the levels of MYC and RNA polymerase II at the ZFP281- and MYC-cobound genes. Specially, we found that recruitment is required for the regulation of the Lin28a oncogene and pri-let-7 transcription. Our results therefore suggest a major role of ZFP281 in recruiting MYC to chromatin and the integration of ZFP281 and the MYC/LIN28A/Let-7 loop into a multilevel circuit.

AFF3-DNA methylation interplay in maintaining the mono-allelic expression pattern of XIST in terminally differentiated cells.

X chromosome inactivation and genomic imprinting are two classic epigenetic regulatory processes that cause mono-allelic gene expression. In female mammals, mono-allelic expression of the long non-coding RNA gene X-inactive specific transcript (XIST) is essential for initiation of X chromosome inactivation upon differentiation. We have previously demonstrated that the central factor of super elongation complex-like 3 (SEC-L3), AFF3, is enriched at gamete differentially methylated regions (DMRs) of the imprinted loci and regulates the imprinted gene expression. Here, we found that AFF3 can also bind to the DMR downstream of the XIST promoter. Knockdown of AFF3 leads to de-repression of the inactive allele of XIST in terminally differentiated cells. In addition, the binding of AFF3 to the XIST DMR relies on DNA methylation and also regulates DNA methylation level at DMR region. However, the KAP1-H3K9 methylation machineries, which regulate the imprinted loci, might not play major roles in maintaining the mono-allelic expression pattern of XIST in these cells. Thus, our results suggest that the differential mechanisms involved in the XIST DMR and gDMR regulation, which both require AFF3 and DNA methylation.

Striking a balance: regulation of transposable elements by Zfp281 and Mll2 in mouse embryonic stem cells.

Transposable elements (TEs) compose about 40% of the murine genome. Retrotransposition of active TEs such as LINE-1 (L1) tremendously impacts genetic diversification and genome stability. Therefore, transcription and transposition activities of retrotransposons are tightly controlled. Here, we show that the Krüppel-like zinc finger protein Zfp281 directly binds and suppresses a subset of retrotransposons, including the active young L1 repeat elements, in mouse embryonic stem (ES) cells. In addition, we find that Zfp281-regulated L1s are highly enriched for 5-hydroxymethylcytosine (5hmC) and H3K4me3. The COMPASS-like H3K4 methyltransferase Mll2 is the major H3K4me3 methylase at the Zfp281-regulated L1s and required for their proper expression. Our studies also reveal that Zfp281 functions partially through recruiting the L1 regulators DNA hydroxymethylase Tet1 and Sin3A, and restricting Mll2 at these active L1s, leading to their balanced expression. In summary, our data indicate an instrumental role of Zfp281 in suppressing the young active L1s in mouse ES cells.

Transcriptional Elongation Control of Hepatitis B Virus Covalently Closed Circular DNA Transcription by Super Elongation Complex and BRD4.

Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. HBV reactivation during or after chemotherapy is a potentially fatal complication for cancer patients with chronic HBV infection. Transcription of HBV is a critical intermediate step of the HBV life cycle. However, factors controlling HBV transcription remain largely unknown. Here, we found that different P-TEFb complexes are involved in the transcription of the HBV viral genome. Both BRD4 and the super elongation complex (SEC) bind to the HBV genome. The treatment of bromodomain inhibitor JQ1 stimulates HBV transcription and increases the occupancy of BRD4 on the HBV genome, suggesting the bromodomain-independent recruitment of BRD4 to the HBV genome. JQ1 also leads to the increased binding of SEC to the HBV genome, and SEC is required for JQ1-induced HBV transcription. These findings reveal a novel mechanism by which the HBV genome hijacks the host P-TEFb-containing complexes to promote its own transcription. Our findings also point out an important clinical implication, that is, the potential risk of HBV reactivation during therapy with a BRD4 inhibitor, such as JQ1 or its analogues, which are a potential treatment for acute myeloid leukemia.

A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron.

Genomic imprinting is an epigenetic regulation that leads to gene expression in a parent-of-origin specific manner. AFF3, the central component of the Super Elongation Complex-like 3 (SEC-L3), is enriched at both the intergenic-differentially methylated region (IG-DMR) and the Meg3 enhancer within the imprinted Dlk1-Dio3 locus to regulate the allele-specific gene expression in this locus. The localization of AFF3 to IG-DMR requires ZFP57. However, how AFF3 functions at the Meg3 enhancer in maintaining allele-specific gene expression remains unclear. Here, we demonstrate that AFF3 is associated with the Krüppel-like zinc finger protein ZFP281 in mouse embryonic stem (ES) cells. ZFP281 recruits AFF3 to the Meg3 enhancer within the imprinted Dlk1-Dio3 locus, thus regulating the allele-specific expression of the Meg3 polycistron. Our genome-wide analyses further identify ZFP281 as a critical factor generally associating with AFF3 at enhancers and functioning together with AFF3 in regulating the expression of a subset of genes. Our study suggests that different zinc finger proteins can recruit AFF3 to different regulatory elements and differentially regulate the function of AFF3 in a context-dependent manner.

Enhancer, epigenetics, and human disease.

Enhancers encode a huge body of information to determine the precise tissue specific gene expression pattern during normal development. Nowadays, enhancers are also considered as key players in directing disease transcriptional program during pathogenesis. New genomic technologies allow the identification, functional characterization and manipulation of enhancers. The advances in the transcriptional enhancer field hold great promise in linking developmental or disease phenotypes to genetic variants and promoting precision medicine.

Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer.

Zic2 is an enhancer-binding factor required for embryonic stem cell specification.

The Zinc-finger protein of the cerebellum 2 (Zic2) is one of the vertebrate homologs of the Drosophila pair-rule gene odd-paired (opa). Our molecular and biochemical studies demonstrate that Zic2 preferentially binds to transcriptional enhancers and is required for the regulation of gene expression in embryonic stem cells. Detailed genome-wide and molecular studies reveal that Zic2 can function with Mbd3/NuRD in regulating the chromatin state and transcriptional output of genes linked to differentiation. Zic2 is required for proper differentiation of embryonic stem cells (ESCs), similar to what has been previously reported for Mbd3/NuRD. Our study identifies Zic2 as a key factor in the execution of transcriptional fine-tuning with Mbd3/NuRD in ESCs through interactions with enhancers. Our study also points to the role of the Zic family of proteins as enhancer-specific binding factors functioning in development.