Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription—from transcription initiation through to export of mRNA from the nucleus—is influenced by the UPS and that all major arms of the system—from the first step in ubiquitin (Ub) conjugation through to the proteasome—are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power.

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration 1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2) 3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens 4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting. Endothelial cells (ECs) in zonated capillaries sustain tissue-specific homeostasis and supply angiocrine factors to guide organ regeneration 1,2. By contrast, maladaptation of ECs contributes to fibrosis and tumour progression 6,7. The mechanism(s) by which ECs acquire adaptive tissue-specific heterogeneity or maladapt within the scarred tissues or tumour microenvironment are unknown. Identifying the molecular determinants of vascular heterogeneity requires the generation of malleable and perfusable vascular networks that are

Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA, into oocytes in somatic cell nuclear transfer embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity. P ioneering nuclear transfer experiments in amphibians have revealed that the cytoplasm of the egg is able to reprogram a differentiated nucleus to the embryonic state (1, 2). The success of somatic cell nuclear transfer (SCNT) to produce cloned animals using enucleated metaphase II (MII) oocytes (3, 4), and, recently, the successful derivation of SCNT human embryonic stem cells (5), have demonstrated that maternal factors in the mature ooplasm are capable and sufficient to reprogram a differentiated cell nucleus to pluripotency. This process is known to involve a series of sequential events including protein exchange between donor nucleus and ooplasm, donor nuclear chromatin remodeling, and pluripotency gene reactivation (6-12). However, maternal factors responsible for this reprogramming process and the underlying mechanism(s) remain largely unknown.Thousands of different maternal proteins and mRNAs have been found in mouse mature oocytes (13,14), including variants of the core histone proteins that, along with DNA, constitute nucleosomes. Accumulating evidence suggests that histone variants play important roles in chromatin remodeling and epigenetic regulation orchestrating gene expression changes during reprogramming (12,15,16). In mammals, the histone variant H3.3 is encoded by two different genes (h3f3a and h3f3b), whose translation results in an identical protein product (17, 18). Unlike canonical H3 histones that are expressed and incorporated into chromatin during S phase, expression of H3.3 is not cell cycle-regulated, and the variant is expressed in quiescent cells, postmitotic cells, and proliferating cells throughout the whole cycle, enabling H3.3 deposition in a DNA synthesis-independent manner during and outside of S phase (19). It has been suggested that matern...

Eucaryotic gene expression requires chromatin-remodeling activities. We show by time-course studies that transcriptional induction of the yeast glucose-regulated SUC2 gene is rapid and shows a striking biphasic pattern, the first phase of which is partly mediated by the general stress transcription factors Msn2p/Msn4p. The SWI/SNF ATP-dependent chromatin-remodeling complex associates with the promoter in a similar biphasic manner and is essential for both phases of transcription. Two different histone acetyltransferases, Gcn5p and Esa1p, enhance the binding of SWI/SNF to the promoter during early transcription and are required for optimal SUC2 induction. Gcn5p is recruited to SUC2 simultaneously with SWI/ SNF, whereas Esa1p associates constitutively with the promoter. This study reveals an unusual transcription pattern of a metabolic gene and suggests a novel strategy by which distinct chromatin remodelers cooperate for the dynamic activation of transcription.

No abstract

Covalent modification of histones by ubiquitylation is a prominent epigenetic mark that features in a variety of chromatin-based events such as histone methylation, gene silencing, and repair of DNA damage. The prototypical example of histone ubiquitylation is that of histone H2B in Saccharomyces cerevisiae. In this case, attachment of ubiquitin to lysine 123 (K123) of H2B is important for regulation of both active and transcriptionally silent genes and participates in trans to signal methylation of histone H3. It is generally assumed that H2B is monoubiquitylated at K123 and that it is this single ubiquitin moiety that influences H2B function. To determine whether this assumption is correct, we have re-examined the ubiquitylation status of endogenous H2B in yeast. We find that, contrary to expectations, H2B is extensively polyubiquitylated. Polyubiquitylation of H2B appears to occur within the context of chromatin and is not associated with H2B destruction. There are at least two distinct modes of H2B polyubiquitylation: one that occurs at K123 and depends on the Rad6 -Bre1 ubiquitylation machinery and another that occurs on multiple lysine residues and is catalyzed by an uncharacterized ubiquitin ligase(s). Interestingly, these ubiquitylation events are under the influence of different combinations of ubiquitin-specific proteases, suggesting that they have distinct biological functions. These results raise the possibility that some of the biological effects of ubiquitylation of H2B are exerted via ubiquitin chains, rather than a single ubiquitin group. INTRODUCTIONOver the last decade, it has become apparent that covalent modification of histones plays a prominent role in establishing epigenetic patterns of gene control in eukaryotic cells (Ruthenburg et al., 2007). A variety of histone modifications have been described, including phosphorylation, methylation, and ubiquitylation. These modifications frequently occur in distinct, interrelated patterns, giving rise to the notion that there is a histone code that is read by the cellular machinery to set the transcriptional status of a particular piece of chromatin (Jenuwein and Allis, 2001).Interestingly, one of the first covalent histone modifications to be discovered was ubiquitylation. In their characterization of the nuclear protein "A24," Busch and colleagues (Goldknopf et al., 1975;Goldknopf and Busch, 1977) described an isopeptide linkage between lysine 119 (K119) of histone H2A and the protein that is now known as ubiquitin (Ub). Early studies (e.g., Levinger and Varshavsky, 1982) demonstrated a connection between histone ubiquitylation and the transcriptional status of chromatin, and it is now clear that histone ubiquitylation, which has been reported for H2A, H2B, H3, and H4 (Muratani and Tansey, 2003), is an important regulatory modification involved in both gene silencing (e.g., de Napoles et al., 2004;Fang et al., 2004) and activation (e.g., Kao et al., 2004). Because of the genetics available in yeast, most of what is known about the functional sign...

Emerging evidence suggests that components of the ubiquitin-proteasome system are involved in the regulation of gene expression. A variety of factors, including transcriptional activators, coactivators, and histones, are controlled by ubiquitylation, but the mechanisms through which this modification can function in transcription are generally unknown. Here, we report that the Saccharomyces cerevisiae protein Asr1 is a RING finger ubiquitin-ligase that binds directly to RNA polymerase II via the carboxyl-terminal domain (CTD) of the largest subunit of the enzyme. We show that interaction of Asr1 with the CTD depends on serine-5 phosphorylation within the CTD and results in ubiquitylation of at least 2 subunits of the enzyme, Rpb1 and Rpb2. Ubiquitylation by Asr1 leads to the ejection of the Rpb4/Rpb7 heterodimer from the polymerase complex and is associated with inactivation of polymerase function. Our data demonstrate that ubiquitylation can directly alter the subunit composition of a core component of the transcriptional machinery and provide a paradigm for how ubiquitin can influence gene activity.transcription ͉ ubiquitin T he correct regulation of gene transcription depends on mechanisms that regulate the formation and dynamics of large multiprotein complexes during various stages of the transcription process. One of the most prominent of these mechanisms is posttranslational protein modification. Phosphorylation is frequently used to promote and stabilize the interaction of various proteins; recruitment of capping and splicing factors to elongating RNA polymerase II (pol II), for example, is signaled by phosphorylation within the carboxyl-terminal domain (CTD) of its largest subunit (1), although modifications such as methylation (2) and acetylation (3) can also influence critical protein-protein interactions. One modification that has received attention in recent years is ubiquitylation, as it has become evident that modification of transcription proteins by ubiquitin (Ub) (4, 5) plays a role in diverse aspects of gene regulation.Ub is a 76-amino acid protein that is covalently linked to other proteins by the action of an enzymatic cascade, the last step of which is mediated via a Ub-protein ligase (or E3) that recognizes a specific element within the substrate and promotes transfer of Ub to a lysine residue(s) within that protein (6). The utility of ubiquitylation stems from its specificity and its ability to function as either a ''classic'' modification or as a signal for substrate destruction by the 26S proteasome. By varying the extent of protein ubiquitylation, the type of poly-Ub chains, or the sites of ubiquitylation in the substrate, Ub can act as either a reversible modifier of protein function or an irreversible mechanism for limiting protein levels.Several recent sets of studies have revealed that ubiquitylation influences multiple steps in the transcription process. Our particular interest has centered on the connection between ubiquitylation of transcriptional activators and the regulation of...