Greenleaf Lab Interests

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Summary

We are investigating mechanisms by which several activities in the cell nucleus are connected to the transcription apparatus via interactions with the hyper-phosphorylated C-terminal repeat domain (PCTD) of elongating RNA polymerase II. First, we are characterizing the patterns of phosphorylation generated on the CTD by CTD Kinase I, the principal elongation-phase CTD kinase in yeast (human homologue = P-TEFb) [Ref. 1, below]. Then, we are identifying numerous factors that interact with this form of the PCTD [Ref. 2]. Our extant results predict a number of novel roles for elongating RNAPII, and they have engendered several new lines of investigation. Ongoing and future efforts promise to illuminate the physical and functional connections between transcription elongation and other nuclear processes, such as chromatin remodeling, DNA damage repair, RNA processing and RNA quality control.

Current research projects address the following questions:

1. What proteins bind directly to the PCTD of elongating RNAPII?
• How is this binding effected?
• What functions or processes are thereby linked to elongating RNAPII?
1. What are the actual patterns of phosphorylation on different repeats along the CTD of elongating RNAPII in vivo?
2. Are bound factors arranged in non-random order along the PCTD?
• In what additional processes does the PCTD play a role?

Details

Current research projects address the following questions:

1. What proteins bind directly to the PCTD of elongating RNAPII?
• How is this binding effected?
• What functions or processes are thereby linked to elongating RNAPII?

Approach: Our recent studies of the histone methyltransferase Set2 [Refs. 3 & 4, below] serve as a paradigm for investigations of additional PCTD-associating proteins (PCAPs). Using recombinant versions of different Set2 domains we showed that a C-terminal segment of just over 100 amino acids binds directly to the PCTD. The binding specificity of this SRI (Set2-RNAP interacting) domain was examined using BIACORE and a series of synthetic CTD repeat peptides carrying SerPO4 residues in different patterns. The SRI domain shows high selectivity for heptad repeats that carry phosphate groups on both Ser2 and Ser5; satisfyingly this is the pattern most efficiently created by CTDK&endash;I [Ref. 1]. The structure of the SRI domain, determined in a collaborative effort with the lab of Pei Zhou in the Department, is a novel 3-helix bundle with the PCTD binding site comprising portions of helices 1 and 2. Mutational and binding analyses pinpointed 5 residues critical for binding and led to a proposal for how Ser2,5-phosphorylated repeats fit into the binding site [Ref. 4]. Determination of the structure of a complex of {SRI domain + phospho-peptide} is underway.

In addition to biochemical and structural studies, we collaborated with the lab of Brian Strahl (University of North Carolina, Chapel Hill) in performing genetic experiments that demonstrated selective loss of Set2-mediated histone H3 methylation when the SRI domain was deleted. The concurrent loss of the ability of Set2 to bind RNAPII revealed the necessity for an intact SRI domain [Ref. 3]. The detailed findings for Set2 support our proposal that PCAPs and associated proteins identified in our search through the proteome tether a variety of nuclear factors, representing numerous functions, to elongating RNAPII (Figs 1 & 4) [cf. Ref. 2].

Experiments

With these studies of Set2 to guide us, we are beginning detailed investigations into additional PCAPs. We are searching through many of the potential PCTD-associating proteins described in [Ref. 2] to identify those that bind directly to the PCTD. From these we will select a small but functionally-diverse subset of the PCAPs to investigate in detail. For each of these proteins we will ask:

• What domain mediates binding?

• What is the phospho-epitope binding specificity of this domain?

• What are the structures of the domain and its binding interface?

• What are the functional consequences of PCTD binding?

• How is binding regulated?

Results of these studies will result in greatly increased understanding of PCTD-mediated nuclear events.

• What are the actual patterns of phosphorylation on different repeats along the CTD of elongating RNAPII in vivo?
  • Are bound factors arranged in non-random order along the PCTD?

It is not known how many of the 26 yeast repeats, or 52 mammalian repeats, are actually phosphorylated in vivo. Also, how the pattern of phosphates on neighboring repeats may differ is not known. These details are extremely important, since the patterns of phosphorylation determine which factors bind the PCTD. To uncover these details, we are engineering altered versions of the CTD into Rpb1, the largest subunit of RNAPII. One set of alterations includes CTDs with cleavage sites placed at intervals along the CTD. These variant Rpb1s will be used to replace the normal Rpb1 in yeast by directed gene replacement. Affinity purification of RNAPII from these strains, followed by cleavage and phospho-amino acid analysis, will produce new insights into phospho-modification patterns in vivo.

The same constructs will enable us to ask if there is a spatial order to PCAP binding along the CTD. Isolation of a particular gene at different moments after induction of transcription (methods under development) will be followed by cleavage, separation of CTD segments, and identification of PCAPs associated with each segment. It will be exciting to determine if there is an non-random arrangement of factors along the CTD. The mechanisms leading to such an outcome will be fascinating to investigate.

PCTD roles in other processes

Identifying Set2 as a PCAP enabled us to show that the PCTD plays an essential role in methylation of Histone H3 on Lys 36. Recognizing that a group of CTD-binding proteins overlap with proteins involved in responses to ionizing radiation (in progress) has led to the realization that the PCTD (made by CTDK-I) is importantly involved in these DNA damage responses. As we continue our studies, we will identify and characterize more direct-binding PCAPs from among the 100 proteins described in Ref. 2. These investigations will provide novel insights into additional roles for the PCTD. Given what we have uncovered so far, these roles promise to be fundamentally important and medically relevant.

References