The human RNA polymerase II-associated factor 1 (hPaf1): a new regulator of cell-cycle progression.

BACKGROUND: The human PAF (hPAF) complex is part of the RNA polymerase II transcription apparatus and regulates multiple steps in gene expression. Further, the yeast homolog of hPaf1 has a role in regulating the expression of a subset of genes involved in the cell-cycle. We therefore investigated the role of hPaf1 during progression of the cell-cycle. METHODOLOGY/FINDINGS: Herein, we report that the expression of hPaf1, a subunit of the hPAF complex, increases with cell-cycle progression and is regulated in a cell-cycle dependant manner. hPaf1 specifically regulates a subclass of genes directly implicated in cell-cycle progression during G1/S, S/G2, and G2/M. In prophase, hPaf1 aligns in filament-like structures, whereas in metaphase it is present within the pole forming a crown-like structure, surrounding the centrosomes. Moreover, hPaf1 is degraded during the metaphase to anaphase transition. In the nucleus, hPaf1 regulates the expression of cyclins A1, A2, D1, E1, B1, and Cdk1. In addition, expression of hPaf1 delays DNA replication but favors the G2/M transition, in part through microtubule assembly and mitotic spindle formation. CONCLUSION/SIGNIFICANCE: Our results identify hPaf1 and the hPAF complex as key regulators of cell-cycle progression. Mutation or loss of stoichiometry of at least one of the members may potentially lead to cancer development

The Human RNA Polymerase II-Associated Factor 1 (hPaf1): A New Regulator of Cell-Cycle Progression

The human PAF (hPAF) complex is part of the RNA polymerase II transcription apparatus and regulates multiple steps in gene expression. Further, the yeast homolog of hPaf1 has a role in regulating the expression of a subset of genes involved in the cell-cycle. We therefore investigated the role of hPaf1 during progression of the cell-cycle.Herein, we report that the expression of hPaf1, a subunit of the hPAF complex, increases with cell-cycle progression and is regulated in a cell-cycle dependant manner. hPaf1 specifically regulates a subclass of genes directly implicated in cell-cycle progression during G1/S, S/G2, and G2/M. In prophase, hPaf1 aligns in filament-like structures, whereas in metaphase it is present within the pole forming a crown-like structure, surrounding the centrosomes. Moreover, hPaf1 is degraded during the metaphase to anaphase transition. In the nucleus, hPaf1 regulates the expression of cyclins A1, A2, D1, E1, B1, and Cdk1. In addition, expression of hPaf1 delays DNA replication but favors the G2/M transition, in part through microtubule assembly and mitotic spindle formation.Our results identify hPaf1 and the hPAF complex as key regulators of cell-cycle progression. Mutation or loss of stoichiometry of at least one of the members may potentially lead to cancer development

Overexpression of PD2 leads to increased tumorigenicity and metastasis in pancreatic ductal adenocarcinoma.

Pancreatic differentiation 2 (PD2), an important subunit of the human PAF complex, was identified after differential screening analysis of 19q13 amplicon, and its overexpression induces oncogenic transformation of NIH3T3 cells, hence raising the possibility of a role for PD2 in tumorigenesis and metastasis. To test this hypothesis, we analyzed here the functional role and clinical significance of PD2 in pancreatic ductal adenocarcinoma (PDAC) and its pathogenesis. Using immunohistochemical analysis, we found that PD2 is detected in the acini but not in the ducts in the normal pancreas. In human PDAC specimens, PD2 was instead primarily detected in the ducts (12/48 patients 25%; p-value \u3c 0.0001), thereby showing that PDAC correlates with increased ductal expression of PD2. Consistently, PD2 expression was increased in telomerase-immortalized human pancreatic ductal cells (HPNE cells) modified to express the HPV16 E6 and E7 proteins, whose respective functions are to block p53 and RB. In addition, ectopic expression of PD2 in PDAC cells (Capan-1 and SW1990) led to increased clonogenicity and migration in vitro, and tumor growth and metastasis in vivo. Interestingly, PD2 overexpression also resulted in enrichment of cancer stem cells (CSCs) and upregulation of oncogenes such as c-Myc and cell cycle progression marker, cyclin D1. Taken together, our results support that PD2 is overexpressed in the ducts of PDAC tissues, and results in tumorigenesis and metastasis via upregulation of oncogenes such as c-Myc and cyclin hence D1 implicating PD2 upregulation in pancreatic oncogenesis with targeted therapeutic potential

Human RNA Polymerase II-Association Factor 1 (hPaf1/PD2) Regulates Histone Methylation and Chromatin Remodeling in Pancreatic Cancer

Change in gene expression associated with pancreatic cancer could be attributed to the variation in histone posttranslational modifications leading to subsequent remodeling of the chromatin template during transcription. However, the interconnected network of molecules involved in regulating such processes remains elusive. hPaf1/PD2, a subunit of the human PAF-complex, involved in the regulation of transcriptional elongation has oncogenic potential. Our study explores the possibility that regulation of histone methylation by hPaf1 can contribute towards alteration in gene expression by nucleosomal rearrangement. Here, we show that knockdown of hPaf1/PD2 leads to decreased di- and tri-methylation at histone H3 lysine 4 residues in pancreatic cancer cells. Interestingly, hPaf1/PD2 colocalizes with MLL1 (Mixed Lineage Leukemia 1), a histone methyltransferase that methylates H3K4 residues. Also, a reduction in hPaf1 level resulted in reduced MLL1 expression and a corresponding decrease in the level of CHD1 (Chromohelicase DNA-binding protein 1), an ATPase dependent chromatin remodeling enzyme that specifically binds to H3K4 di and trimethyl marks. hPaf1/PD2 was also found to interact and colocalize with CHD1 in both cytoplasmic and nuclear extracts of pancreatic cancer cells. Further, reduced level of CHD1 localization in the nucleus in hPaf1/PD2 Knockdown cells could be rescued by ectopic expression of hPaf1/PD2. Micrococcal nuclease digestion showed an altered chromatin structure in hPaf1/PD2-KD cells. Overall, our results suggest that hPaf1/PD2 in association with MLL1 regulates methylation of H3K4 residues, as well as interacts and regulates nuclear shuttling of chromatin remodeling protein CHD1, facilitating its function in pancreatic cancer cells

hPaf1 during cell cycle progression.

<p>hPaf1 expression initiates in early G1, increases during the S-phase and reaches a maximum level in early G2. Synthesized as a 60 kDa protein, hPaf1 translocates as an 80 kDa protein into the nucleus. In the nucleus, hPaf1 directly regulates the expression of the cyclins A1, A2, D1, E1, B1. In S phase, hPaf1 interacts with the DNA polymerase α-primase complex and delays the onset of DNA replication. At mitosis, hPaf1 migrates to the cellular pole, forming a crown surrounding the centrosomes. It is then possibly shuttled via tubulin filaments to be degraded by the proteasome.</p

hPaf1 is a key regulator for temporal transcription of cyclins.

<p>A) Chromatine immunoprecipitation using hPaf1 anitbody for cyclins A1, A2, E1, D1, B1, and the Cdk1. An aliquot of the DNA extract before immunoprecipitation was used as a positive control for the PCR amplification. B) Analysis of cyclins E1, B1, D1, and A2 expression was done by relative competitive dropping-PCR on T0 and T+8 synchronized Panc1 cells. To measure the effect of hPaf1 expression, hPaf1 was down-regulated using the siRNA technology. C) Values of the dropping-PCR were adjusted for assay variation by dividing the integrated optical density of cyclins A2, B1, D1, and E1 mRNA by the integrated optical density of GAPDH mRNA. D) Suppression of hPaf1 expression by the Panc1 cells after transfection with siRNA oligonucleotide was measured on the same RNAs used for the dropping-PCR by one-step real time RT-PCR using a TaqMan probe following the absolute standard curve method. Standard curves were established using hPaf1 and GAPDH mimics. Sequences and conditions used in this experiment are available upon request.</p

Localization of hPaf1 during the cell cycle in Panc1 cells.

<p>hPaf1 expression was monitored in a synchronously growing population of Panc1 cells. Panc1 cells were arrested at the G1/S boundary by double thymidine block, then released in normal medium containing serum (time 0 = T0) and analyzed every 2 h for 24 h by flow cytometry (after propidium iodide staining), confocal microscopy, and Western blotting of cytoplasmic and nuclear cell fractions. A) Eighty percent of the cells were blocked at the G1/S boundary. After release, the cells progressed into the cell cycle synchronously to reach a peak of mitosis after 10–12 h. Representative fields from the confocal analysis are presented in B). The cells were grown at low density on cover slips for 24 h, and arrested at the G1/S boundary by double thymidine incorporation. The acetone/methanol (1∶1)-fixed Panc1 cells were labeled using a FITC-conjugated anti-hPaf1 monoclonal antibody and counterstained by propidium iodide. The amount of fluorescence detected for the cells in T+12 (G2/M) was twice the level detected in T0 (G1/S). Up to 10 fields were monitored for each phase of the cell cycle. Cells in the prophase, metaphase, anaphase, and telophase showed very low to no expression of hPaf1. C) Nuclear and cytoplasmic protein extracts were prepared every 2 h and analyzed by immunoblotting using anti-hPaf1 and anti-hLeo1 antibodies. The JLA-20 antibody recognizing β actin was used as a loading control.</p