The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.

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Background-We sought to identify preoperative predictors of clinical outcomes after surgery in patients with severe tricuspid regurgitation. Methods and Results-We prospectively enrolled 61 consecutive patients (54 women, aged 57Ϯ9 years) with isolated severe tricuspid regurgitation undergoing corrective surgery. Twenty-one patients (34%) were in New York Heart Association functional class II, 35 (57%) in class III, and 5 (9%) in class IV. Fifty-seven patients (93%) had previous history of left-sided valve surgery. Preoperative echocardiography revealed pulmonary artery systolic pressure of 41.5Ϯ8.7 mm Hg, right ventricular (RV) end-diastolic area of 35.1Ϯ9.0 cm

Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT hybrid regulator contains binding sites for several nuclear proteins, including two sites for PTF1 (9,19,38).In this paper, we demonstrate that the hybrid construct containing the amylase/elastase regulator fused to the selectable GPT gene is not expressed after transfection into mouse hepatoma or fibroblast cell lines but can be activated by fusion with pancreatic acinar cell lines. The nonexpressed construct is thus present in a state which permits activation by trans-acting factors. MATERIALS AND METHODSPlasmid construction. An amylase/elastase/CAT construct (AmyEICAT) (19) containing the Amy2.2 enhancer (bp -208 to -82) and the rat elastase I enhancer/promoter (bp -205 to +8) was digested with HindIll and BamHI to yield the hybrid enhancer/promoter fragment. This hybrid enhancer/ promoter element was used to replace the simian virus 40 (SV40) promoter region in pSV2GPT (27,28,37) by the following protocol. pSV2GPT was digested with AccI, blunt ended, ligated with HindIII linkers, and digested with BglII to remove the SV40 enhancer and promoter elements. AmyEIGPT was made by directional cloning in which the HindIII-BamHI fragment from AmyEICAT was ligated to the promoterless GPT construct (Fig. 1). EIGPT was made by subcloning the HindIII-BamHI fragment from EICAT (23) which contains the rat elastase I enhancer/promoter (bp -205 to +8) into the same promoterless GPT-containing vector. pG2EIGPT, used for the detection of GPT transcripts, was generated by subcloning the HindIII-KpnI fragment of EIGPT (bp -208