TAP-MS analysis of FACT interactions and regulation by a ubiquitin ligase, San1.

An evolutionarily conserved heterodimeric FACT (Facilitates chromatin transcription) regulates transcription, DNA repair, replication and other cellular processes via its interactions with other proteins. FACT is recently found to be regulated via ubiquitylation and 26S proteasomal degradation, alteration of which is associated with aberrant transcription and genome integrity. However, there has not been a systematic study to analyze FACT interactions proteome-wide in the presence and absence of its UPS (Ubiquitin-proteasome system) regulation, which could reveal new FACT interactors with mechanistic and functional implications.

Ubiquitin-proteasome system regulation of a key gene regulatory factor, Paf1C.

Paf1 (Polymerase-associated factor 1) complex (Paf1C) is evolutionarily conserved from yeast to humans, and facilitates transcription elongation as well as co-transcriptional histone covalent modifications and mRNA 3'-end processing. Thus, Paf1C is a key player in regulation of eukaryotic gene expression. Paf1C consists of Paf1, Cdc73, Ctr9, Leo1 and Rtf1 in both yeast and humans, but it has an additional component, Ski8, in humans.

Chromatin and non-chromatin immunoprecipitations to capture protein-protein and protein-nucleic acid interactions in living cells.

Proteins are expressed from genes via sequential biological processes of transcription, mRNA processing, export and translation, and play their roles in maintaining cellular functions via interactions with proteins, DNAs or RNAs. Thus, it is important to study the protein interactions during biological processes in living cells towards understanding their mechanisms-of-action in real time. Methodologies have been developed over the years to study protein interactions in vivo.

Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins.

Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex.

A novel ubiquitin-proteasome system regulation of Sgf73/ataxin-7 that maintains the integrity of the coactivator SAGA in orchestrating transcription.

Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation.

Genome-Wide Regulations of the Preinitiation Complex Formation and Elongating RNA Polymerase II by an E3 Ubiquitin Ligase, San1.

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in the genome-wide association of TATA box binding protein (TBP; which nucleates preinitiation complex [PIC] formation for transcription initiation) and RNA polymerase II (Pol II).

Proteasomal Regulation of Mammalian SPT16 in Controlling Transcription.

FACT (cilitates hromatin ranscription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer.

An F-Box Protein, Mdm30, Interacts with TREX Subunit Sub2 To Regulate Cellular Abundance Cotranscriptionally in Orchestrating mRNA Export Independently of Splicing and Mitochondrial Function.

Although an F-box protein, Mdm30, is found to regulate ubiquitylation of the Sub2 component of TREX (anscription-port) complex for proteasomal degradation in stimulation of mRNA export, it remains unknown whether such ubiquitin-proteasome system (UPS) regulation of Sub2 occurs cotranscriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in the absence of Mdm30 alters mRNA export via splicing defects of export factors and/or mitochondrial dynamics/function, since Sub2 controls mRNA splicing and Mdm30 regulates mitochondrial aggregation. Here, we show that Mdm30 interacts with Sub2, and temporary shutdown of Mdm30 enhances Sub2's abundance and impairs mRNA export.

Distinct Functions of the Cap-Binding Complex in Stimulation of Nuclear mRNA Export.

Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5' end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export.

TOR Facilitates the Targeting of the 19S Proteasome Subcomplex To Enhance Transcription Complex Assembly at the Promoters of the Ribosomal Protein Genes.

TOR (target of rapamycin) has been previously implicated in transcriptional stimulation of the ribosomal protein (RP) genes via enhanced recruitment of NuA4 (nucleosome acetyltransferase of H4) to the promoters. However, it is not clearly understood how TOR enhances NuA4 recruitment to the promoters of the RP genes. Here we show that TOR facilitates the recruitment of the 19S proteasome subcomplex to the activator to enhance the targeting of NuA4 to the promoters of the RP genes.

Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling.

SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of , a gene for the high-affinity inorganic phosphate (P) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of occurs predominantly in a TFIID-dependent manner in the absence of P in the growth medium.

Ubiquitin-Proteasome System Regulation of an Evolutionarily Conserved RNA Polymerase II-Associated Factor 1 Involved in Pancreatic Oncogenesis.

The evolutionarily conserved RNA polymerase II-associated factor 1 (Paf1) from yeast to humans regulates transcription and associated processes, and thus, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Indeed, Paf1 (also known as PD2 or pancreatic differentiation 2) is found to be upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown.

An mRNA Capping Enzyme Targets FACT to the Active Gene To Enhance the Engagement of RNA Polymerase II into Transcriptional Elongation.

We have recently demonstrated that an mRNA capping enzyme, Cet1, impairs promoter-proximal accumulation/pausing of RNA polymerase II (Pol II) independently of its capping activity in to control transcription. However, it is still unknown how Pol II pausing is regulated by Cet1. Here, we show that Cet1's N-terminal domain (NTD) promotes the recruitment of FACT (cilitates hromatin ranscription that enhances the engagement of Pol II into transcriptional elongation) to the coding sequence of an active gene, , independently of mRNA-capping activity.

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation.

FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine-tuned for normal cellular functions. Here, we show that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome.

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' 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.