Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo.

NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a Δeaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions

Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors.

NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3\u27 end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3\u27 end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription

REGULATION OF EUKARYOTIC TRANSCRIPTIONAL ELONGATION AND ASSOCIATED DNA REPAIR

Transcriptional elongation is a crucial step in eukaryotic gene regulation whose mis-regulation leads to cellular pathologies. This makes it quite imperative to aim for a better understanding of the processes regulating transcriptional elongation. An important process promoting the association of RNA Polymerase II (RNAPII) with the coding region of the active gene and hence transcriptional elongation is the monoubiquitination of histone H2B at lysine 123. A complex of an E2 conjugase, Rad6p, and an E3 ligase, Bre1p, is essential for this process. Consistent with the role of histone H2B monoubiquitination in promoting the association of RNAPII with the active gene, this process was found to be impaired in the absence of Rad6p or point mutation of lysine 123 to arginine (H2B-K123R). Intriguingly, the association of RNAPII with the coding region of the active gene was not impaired in the absence of Bre1p, even though Bre1p is essential for histone H2B monoubiquitination. However, deletion of Bre1p’s RING domain that is essential for histone H2B monoubiquitination led to an impaired RNAPII association with the active gene. This observation indicates a role of the non-RING domain of Bre1p in repressing the association of RNAPII with the active gene, resulting in no net decrease in RNAPII occupancy in the absence of Bre1p. Taken together, my results implicated both the stimulatory and repressive roles of the histone H2B ubiquitin ligase Bre1p in regulation of RNAPII association with the coding regions of active genes and hence transcriptional elongation. Interestingly, my work also revealed that for efficient transcriptional elongation by histone H2B monoubiquitination, its optimum level needs to be maintained by a proper balance between Rad6p-Bre1p-mediated ubiquitination and de-ubiquitination (DUB) by the DUB module of SAGA. It was found that Sus1p, a subunit of the DUB module, promotes transcriptional elongation, DNA repair and replication via regulation of histone H2B DUB. In addition to Rad6p- Bre1p and the DUB module, global level of histone H2B monoubiquitination is also critically regulated by Cdk9, a kinase essential for phosphorylation of the serine 2 residue in the C-terminal domain (CTD) of RNAPII, which promotes transcriptional elongation. Apart from serine phosphorylation, proline residues at RNAPII-CTD undergo isomerization by proline isomerases, which also regulate transcription. One of the proline isomerases, Rrd1p, has been previously implicated in transcription in response to rapamycin treatment. Based on this fact and Rrd1p’s known interaction with RNAPII-CTD, we predicted that Rrd1p might regulate transcription independently of rapamycin treatment. In agreement with this hypothesis, our work revealed Rrd1p’s role in facilitating transcription of both rapamycin responsive and non-responsive genes in the absence of rapamycin treatment. Consistently, the absence of Rrd1p led to an impaired nucleosomal disassembly at the active gene, which correlates with the role of Rrd1p in promoting transcription. This is because maintenance of proper nucleosomal dynamics is essential for efficient transcription. It is known that transcriptional elongation is facilitated by the regulation of nucleosomal dynamics via the histone chaperone, FACT. Efficient chromatin reassembly in the wake of elongating RNAPII contributing to the fidelity of transcription is promoted by FACT. Being evolutionarily conserved among eukaryotes, FACT is also known to regulate DNA replication and repair, apart from transcription. Intriguingly, FACT has been found to be upregulated in cancers while its downregulation leads to tumor cell death. However, the mechanism which fine-tunes FACT for normal cellular functions remained unknown. My studies revealed a novel mechanism of regulation of FACT by the ubiquitin-proteasome system in yeast. San1p, an E3 ligase involved in nuclear protein quality control, was found to associate with the active gene and regulate transcriptional elongation through its E3 ligase activity- mediated turnover of Spt16p component of FACT. This regulation was found to maintain optimum level of Spt16p/FACT to engage with the active gene for proper transcriptional elongation, DNA repair and replication. In spite of playing such crucial roles in gene regulation, it was not known how FACT is targeted to the active gene. We discovered that a direct physical interaction between FACT and Cet1p, the mRNA capping enzyme, targets FACT to the active gene independently of Cet1p’s mRNA capping activity. Such targeting of FACT to the active gene leads to the release of promoter proximally paused-RNAPII into transcriptional elongation. However, the progress of RNAPII along the active gene during transcriptional elongation is frequently impeded by various kinds of damages along the underlying template DNA. Even though some of these lesions are co-transcriptionally repaired, it was not known whether the repair of extremely toxic DNA double-strand breaks (DSBs) was coupled to transcription. My results showed that DSBs at the transcriptionally active state of a gene are repaired faster than at the inactive state but such repair was not mediated by a co-transcriptional recruitment of DSB repair factors. This observation is in contrast to other DNA repair pathways such as nucleotide excision repair (NER) where repair factors are co-transcriptionally recruited to the lesion containing DNA. In this regard, we found that an NER factor, Rad14p, co-transcriptionally associates with the active gene in the absence of DNA damage to promote transcription, which unraveled a new role of Rad14p in transcription in addition its established role in NER. In summary, my results provide significant novel insights into the regulation of transcriptional elongation and associated processes leading to better understanding of eukaryotic gene expression

Insights into Cardiovascular Defects and Cardiac Epigenome in the Context of COVID-19

Although few in number, studies on epigenome of the heart of COVID-19 patients show that epigenetic signatures such as DNA methylation are significantly altered, leading to changes in expression of several genes. It contributes to pathogenic cardiac phenotypes of COVID-19, e.g., low heart rate, myocardial edema, and myofibrillar disarray. DNA methylation studies reveal changes which likely contribute to cardiac disease through unknown mechanisms. The incidence of severe COVID-19 disease, including hospitalization, requiring respiratory support, morbidity, and mortality, is disproportionately higher in individuals with co-morbidities. This poses unprecedented strains on the global healthcare system. While their underlying conditions make patients more susceptible to severe COVID-19 disease, strained healthcare systems, lack of adequate support, or sedentary lifestyles from ongoing lockdowns have proved detrimental to their underlying health conditions, thus pushing them to severe risk of congenital heart disease (CHD) itself. Prophylactic vaccines against COVID-19 have ushered new hope for CHD. A common connection between COVID-19 and CHD is SARS-CoV-2’s host receptor ACE2, because ACE2 regulates and protects organs, including the heart, in various ways. ACE2 is a common therapeutic target against cardiovascular disease and COVID-19 which damages organs. Hence, this review explores the above regarding CHDs, cardiovascular damage, and cardiac epigenetics, in COVID-19 patients

Do Transgenerational Epigenetic Inheritance and Immune System Development Share Common Epigenetic Processes?

Epigenetic modifications regulate gene expression for development, immune response, disease, and other processes. A major role of epigenetics is to control the dynamics of chromatin structure, i.e., the condensed packaging of DNA around histone proteins in eukaryotic nuclei. Key epigenetic factors include enzymes for histone modifications and DNA methylation, non-coding RNAs, and prions. Epigenetic modifications are heritable but during embryonic development, most parental epigenetic marks are erased and reset. Interestingly, some epigenetic modifications, that may be resulting from immune response to stimuli, can escape remodeling and transmit to subsequent generations who are not exposed to those stimuli. This phenomenon is called transgenerational epigenetic inheritance if the epigenetic phenotype persists beyond the third generation in female germlines and second generation in male germlines. Although its primary function is likely immune response for survival, its role in the development and functioning of the immune system is not extensively explored, despite studies reporting transgenerational inheritance of stress-induced epigenetic modifications resulting in immune disorders. Hence, this review draws from studies on transgenerational epigenetic inheritance, immune system development and function, high-throughput epigenetics tools to study those phenomena, and relevant clinical trials, to focus on their significance and deeper understanding for future research, therapeutic developments, and various applications

Developmental Aspects of SARS-CoV-2, Potential Role of Exosomes and Their Impact on the Human Transcriptome

With over 4.8 million deaths within 2 years, time is of the essence in combating COVID-19. The infection now shows devastating impacts on the younger population, who were not previously predicted to be vulnerable, such as in the older population. COVID-19-related complications have been reported in neonates whose mothers were infected with SARS-CoV-2 during pregnancy, and in children who get infected. Hence, a deeper understanding of the pathophysiology of COVID-19 during various developmental stages and placental transmission is essential. Although a connection has not yet been established between exosomal trafficking and the placental transmission of COVID-19, reports indicate that SARS-CoV-2 components may be trafficked between cells through exosomes. As the infection spreads, the transcriptome of cells is drastically perturbed, e.g., through the severe upregulation of several immune-related genes. Consequently, a major outcome of COVID-19 is an elevated immune response and the detection of viral RNA transcripts in host tissue. In this direction, this review focuses on SARS-CoV-2 virology, its in utero transmission from infected pregnant mothers to fetuses, SARS-CoV-2 and exosomal cellular trafficking, transcriptomic impacts, and RNA-mediated therapeutics against COVID-19. Future research will establish stronger connections between the above processes to develop diagnostic and therapeutic solutions towards COVID-19 and similar viral outbreaks

Advances in understanding epigenetic impacts on dendritic cell regulation and function

Abstract Dendritic cells (DCs) are the only cells empowered with inducing primary immune response among resting naïve T lymphocytes, hence it is crucial to understand the regulation and function of DCs. During an adaptive immune response, DCs acquire antigens from invasive entities and present the antigens on their own cell surface, hence they are also known as professional antigen‐presenting cells. Epigenetic modifications to the genome play an important role in both the development and the function of DCs. In this direction, significant advancements have been made using high‐throughput methods like ATAC‐seq, ChIP‐seq, RNA‐seq, bisulfite‐seq and so forth to uncover chromatin accessibility landscape, genome‐wide transcription factor (TF) binding, complete transcriptomic profiles, and DNA methylation, respectively. DCs lineage specification and function are determined by TF binding, which is dependent on epigenetic modifications. However, major gaps in knowledge still exist regarding how and why aberrant epigenetic modifications result in defective development and function of DCs leading to an impaired immune system. Hence, this review compiles up‐to‐date literature regarding the exquisite regulation of DCs via epigenetic regulation, to emphasize the potential of further epigenetics research on DCs for addressing current gaps of knowledge in the field. The review also briefly highlights recent findings on DC defects in coronavirus disease 2019 revealed by single‐cell RNA‐seq, and the importance of DCs in clinical trials. Overall, the review highlights how epigenetics‐based state‐of‐the‐art high‐throughput technology can help discoveries on DCs which are crucial to the immune system and pathogenesis, and how DCs are currently targeted for therapeutic developments and clinical trials

Kat2a and Kat2b Acetyltransferase Activity Regulates Craniofacial Cartilage and Bone Differentiation in Zebrafish and Mice

Cranial neural crest cells undergo cellular growth, patterning, and differentiation within the branchial arches to form cartilage and bone, resulting in a precise pattern of skeletal elements forming the craniofacial skeleton. However, it is unclear how cranial neural crest cells are regulated to give rise to the different shapes and sizes of the bone and cartilage. Epigenetic regulators are good candidates to be involved in this regulation, since they can exert both broad as well as precise control on pattern formation. Here, we investigated the role of the histone acetyltransferases Kat2a and Kat2b in craniofacial development using TALEN/CRISPR/Cas9 mutagenesis in zebrafish and the Kat2ahat/hat (also called Gcn5) allele in mice. kat2a and kat2b are broadly expressed during embryogenesis within the central nervous system and craniofacial region. Single and double kat2a and kat2b zebrafish mutants have an overall shortening and hypoplastic nature of the cartilage elements and disruption of the posterior ceratobranchial cartilages, likely due to smaller domains of expression of both cartilage- and bone-specific markers, including sox9a and col2a1, and runx2a and runx2b, respectively. Similarly, in mice we observe defects in the craniofacial skeleton, including hypoplastic bone and cartilage and altered expression of Runx2 and cartilage markers (Sox9, Col2a1). In addition, we determined that following the loss of Kat2a activity, overall histone 3 lysine 9 (H3K9) acetylation, the main epigenetic target of Kat2a/Kat2b, was decreased. These results suggest that Kat2a and Kat2b are required for growth and differentiation of craniofacial cartilage and bone in both zebrafish and mice by regulating H3K9 acetylation