John T. Lis

Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters

RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on ∼30% of human genes, transcription extends beyond pre-messenger RNA 3′ cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.

Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans

Recent years have witnessed a sea change in our understanding of transcription regulation: whereas traditional models focused solely on the events that brought RNA polymerase II (Pol II) to a gene promoter to initiate RNA synthesis, emerging evidence points to the pausing of Pol II during early elongation as a widespread regulatory mechanism in higher eukaryotes. Current data indicate that pausing is particularly enriched at genes in signal-responsive pathways. Here the evidence for pausing of Pol II from recent high-throughput studies will be discussed, as well as the potential interconnected functions of promoter-proximally paused Pol II.

Breaking barriers to transcription elongation.

Hundreds of protein factors participate in transcription and its regulation in eukaryotes. Many of these proteins regulate specific genes by targeting upstream promoter regions, whereas a smaller but mechanistically diverse set of factors functions at most genes during RNA polymerase II (Pol II) elongation. These elongation factors can affect mRNA production at particular stages and in different ways during transcription. Some factors act directly on Pol II, whereas others manipulate the chromatin environment.

The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged

Protein-DNA cross-linking of cultured Drosophila cells has shown that, in vivo, prior to the induction of heat shock, there is approximately one molecule of RNA polymerase II associated with the promoter region of the major heat shock gene, hsp70. Here, we show that this promoter-associated RNA polymerase II molecule is transcriptionally engaged and has formed a nascent RNA chain of approximately 25 nucleotides in length, but is apparently arrested at that point and unable to penetrate further into the hsp70 gene without heat induction. The detection of a post-initiation RNA polymerase complex on the promoter region of the inactive gene suggests that there is a transcriptional control mechanism that acts at a step early in transcript elongation.

PARP Goes Transcription

PARP-1, an enzyme that catalyzes the attachment of ADP ribose units to target proteins, plays at least two important roles in transcription regulation. First, PARP-1 modifies histones and creates an anionic poly(ADPribose) matrix that binds histones, thereby promoting the decondensation of higher-order chromatin structures. Second, PARP-1 acts as a component of enhancer/promoter regulatory complexes. Recent studies have shown that both of these activities are critical for gene regulation in vivo.

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of Histone H4 and Is Associated with Silent Chromatin

We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Defining mechanisms that regulate RNA polymerase II transcription in vivo

In the eukaryotic genome, the thousands of genes that encode messenger RNA are transcribed by a molecular machine called RNA polymerase II. Analysing the distribution and status of RNA polymerase II across a genome has provided crucial insights into the long-standing mysteries of transcription and its regulation. These studies identify points in the transcription cycle where RNA polymerase II accumulates after encountering a rate-limiting step. When coupled with genome-wide mapping of transcription factors, these approaches identify key regulatory steps and factors and, importantly, provide an understanding of the mechanistic generalities, as well as the rich diversities, of gene regulation.

NAD^+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1

PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. Herein, we describe previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed chromatin structures. PARP-1 binds in a specific manner to nucleosomes and modulates chromatin structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of chromatin and a modulator of chromatin structure through its intrinsic enzymatic activity.

Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA

The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.

Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit

The minimal DNA sequence required for the formation of a stable complex with Drosophila heat shock factor (HSF) in vitro is an inverted repeat of a 5 bp recognition unit, -GAA-. Surprisingly, both permutations of this 5 bp unit, head-to-head and tail-to-tail, bind to HSF with similar affinity and with striking 2-fold symmetry. HSF also binds to longer arrays of inverted 5 bp units, and the size of the HSF footprint increases with the addition of each 5 bp unit to these arrays. However, the electrophoretic mobility of the HSF-DNA complexes decreases most distinctly with the addition of every three 5 bp units. Cross-linking of purified HSF in the absence of DNA generates complexes with the sizes expected of HSF trimers. We propose that trimers of HSF bind to DNA and that the number of HSF subunits in direct contact with DNA is determined by the number of correctly positioned 5 bp recognition units.