Alessandro Gardini

Long noncoding RNAs with enhancer-like function in human cells

While the long noncoding RNAs (ncRNAs) constitute a large portion of the mammalian transcriptome, their biological functions has remained elusive. A few long ncRNAs that have been studied in any detail silence gene expression in processes such as X-inactivation and imprinting. We used a GENCODE annotation of the human genome to characterize over a thousand long ncRNAs that are expressed in multiple cell lines. Unexpectedly, we found an enhancer-like function for a set of these long ncRNAs in human cell lines. Depletion of a number of ncRNAs led to decreased expression of their neighboring protein-coding genes, including the master regulator of hematopoiesis, SCL (also called TAL1), Snai1 and Snai2. Using heterologous transcription assays we demonstrated a requirement for the ncRNAs in activation of gene expression. These results reveal an unanticipated role for a class of long ncRNAs in activation of critical regulators of development and differentiation.

Integrator mediates the biogenesis of enhancer RNAs

Integrator is a multi-subunit complex stably associated with the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). Integrator is endowed with a core catalytic RNA endonuclease activity, which is required for the 3′-end processing of non-polyadenylated, RNAPII-dependent, uridylate-rich, small nuclear RNA genes. Here we examine the requirement of Integrator in the biogenesis of transcripts derived from distal regulatory elements (enhancers) involved in tissue- and temporal-specific regulation of gene expression in metazoans. Integrator is recruited to enhancers and super-enhancers in a stimulus-dependent manner. Functional depletion of Integrator subunits diminishes the signal-dependent induction of enhancer RNAs (eRNAs) and abrogates stimulus-induced enhancer–promoter chromatin looping. Global nuclear run-on and RNAPII profiling reveals a role for Integrator in 3′-end cleavage of eRNA primary transcripts leading to transcriptional termination. In the absence of Integrator, eRNAs remain bound to RNAPII and their primary transcripts accumulate. Notably, the induction of eRNAs and gene expression responsiveness requires the catalytic activity of Integrator complex. We propose a role for Integrator in biogenesis of eRNAs and enhancer function in metazoans.

PAF1, a Molecular Regulator of Promoter-Proximal Pausing by RNA Polymerase II.

The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol IIs C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II.

AML1/ETO Oncoprotein Is Directed to AML1 Binding Regions and Co-Localizes with AML1 and HEB on Its Targets

A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein.

Integrator regulates transcriptional initiation and pause release following activation.

In unicellular organisms, initiation is the rate-limiting step in transcription; in metazoan organisms, the transition from initiation to productive elongation is also important. Here, we show that the RNA polymerase II (RNAPII)-associated multiprotein complex, Integrator, plays a critical role in both initiation and the release of paused RNAPII at immediate early genes (IEGs) following transcriptional activation by epidermal growth factor (EGF) in human cells. Integrator is recruited to the IEGs in a signal-dependent manner and is required to engage and recruit the super elongation complex (SEC) to EGF-responsive genes to allow release of paused RNAPII and productive transcription elongation.

The many faces of long noncoding RNAs.

Over the past few years, the field of noncoding RNAs has grown from a niche for geneticists into a prominent domain of mainstream biology. Advances in genomic technologies have provided a more comprehensive view of the mammalian genome, improving our knowledge of regions of the genome devoid of protein‐coding potential. A large body of evidence supports the proposal that noncoding RNAs account for a large proportion of the transcriptional output of any given cell and tissue type. This review will delve into the biogenesis and function of long noncoding RNAs. We will discuss our current understanding of these molecules as major chromatin players, and explore future directions in the field.

BET inhibitors suppress ALDH activity by targeting ALDH1A1 super-enhancer in ovarian cancer

The emergence of tumor cells with certain stem-like characteristics, such as high aldehyde dehydrogenase (ALDH) activity due to ALDH1A1 expression, contributes to chemotherapy resistance and tumor relapse. However, clinically applicable inhibitors of ALDH activity have not been reported. There is evidence to suggest that epigenetic regulation of stem-related genes contributes to chemotherapy efficacy. Here, we show that bromodomain and extraterminal (BET) inhibitors suppress ALDH activity by abrogating BRD4-mediated ALDH1A1 expression through a super-enhancer element and its associated enhancer RNA. The clinically applicable small-molecule BET inhibitor JQ1 suppressed the outgrowth of cisplatin-treated ovarian cancer cells both in vitro and in vivo Combination of JQ1 and cisplatin improved the survival of ovarian cancer-bearing mice in an orthotopic model. These phenotypes correlate with inhibition of ALDH1A1 expression through a super-enhancer element and other stem-related genes in promoter regions bound by BRD4. Thus, targeting the BET protein BRD4 using clinically applicable small-molecule inhibitors, such as JQ1, is a promising strategy for targeting ALDH activity in epithelial ovarian cancer. Cancer Res; 76(21); 6320-30. ©2016 AACR.

Genome‐wide analysis reveals a role for BRCA1 and PALB2 in transcriptional co‐activation

Breast and ovarian cancer susceptibility genes BRCA1 and PALB2 have enigmatic roles in cellular growth and mammalian development. While these genes are essential for growth during early developmental programs, inactivation later in adulthood results in increased growth and formation of tumors, leading to their designation as tumor suppressors. We performed genome‐wide analysis assessing their chromatin residence and gene expression responsiveness using high‐throughput sequencing in breast epithelial cells. We found an intimate association between BRCA1 and PALB2 chromatin residence and genes displaying high transcriptional activity. Moreover, our experiments revealed a critical role for BRCA1 and, to a smaller degree, PALB2 in transcriptional responsiveness to NF‐κB, a crucial mediator of growth and inflammatory response during development and cancer. Importantly, we also uncovered a vital role for BRCA1 and PALB2 in response to retinoic acid (RA), a growth inhibitory signal in breast cancer cells, which may constitute the basis for their tumor suppressor activity. Taken together, our results highlight an important role for these breast cancer proteins in the regulation of diverse growth regulatory pathways.

Global Run-On Sequencing (GRO-Seq)

Transcription occurring at gene loci results in accumulation of mature RNA molecules (i.e., mRNAs) that can be easily assayed by RT-PCR or RNA sequencing. However, the steady-state level of RNA does not accurately mirror transcriptional activity per se. In fact, RNA stability plays a major role in determining the relative abundance of any given RNA molecule. Here, I describe a protocol of Nuclear Run-On assay coupled to deep sequencing to assess real-time transcription from engaged RNA polymerase. Mapping nascent transcripts at the genome-wide scale provides a reliable measure of transcriptional activity in mammalian cells and delivers a high-resolution map of coding and noncoding transcripts that is especially useful for annotation and quantification of short-lived RNA molecules.

Requirement for SNAPC1 in transcriptional responsiveness to diverse extracellular signals.

ABSTRACT Initiation of transcription of RNA polymerase II (RNAPII)-dependent genes requires the participation of a host of basal transcription factors. Among genes requiring RNAPII for transcription, small nuclear RNAs (snRNAs) display a further requirement for a factor known as snRNA-activating protein complex (SNAPc). The scope of the biological function of SNAPc and its requirement for transcription of protein-coding genes has not been elucidated. To determine the genome-wide occupancy of SNAPc, we performed chromatin immunoprecipitation followed by high-throughput sequencing using antibodies against SNAPC4 and SNAPC1 subunits. Interestingly, while SNAPC4 occupancy was limited to snRNA genes, SNAPC1 chromatin residence extended beyond snRNA genes to include a large number of transcriptionally active protein-coding genes. Notably, SNAPC1 occupancy on highly active genes mirrored that of elongating RNAPII extending through the bodies and 3′ ends of protein-coding genes. Inhibition of transcriptional elongation resulted in the loss of SNAPC1 from the 3′ ends of genes, reflecting a functional association between SNAPC1 and elongating RNAPII. Importantly, while depletion of SNAPC1 had a small effect on basal transcription, it diminished the transcriptional responsiveness of a large number of genes to two distinct extracellular stimuli, epidermal growth factor (EGF) and retinoic acid (RA). These results highlight a role for SNAPC1 as a general transcriptional coactivator that functions through elongating RNAPII.