Saccharomyces cerevisiae HMO1 interacts with TFIID and participates in start site selection by RNA polymerase II

Abstract

Saccharomyces cerevisiae HMO1, a high mobility group B (HMGB) protein, associates with the rRNA locus and with the promoters of many ribosomal protein genes (RPGs). Here, the Sos recruitment system was used to show that HMO1 interacts with TBP and the N-terminal domain (TAND) of TAF1, which are integral components of TFIID. Biochemical studies revealed that HMO1 copurifies with TFIID and directly interacts with TBP but not with TAND. Deletion of HMO1 (Δhmo1) causes a severe cold-sensitive growth defect and decreases transcription of some TAND-dependent genes. Δhmo1 also affects TFIID occupancy at some RPG promoters in a promoter-specific manner. Interestingly, over-expression of HMO1 delays colony formation of taf1 mutants lacking TAND (taf1ΔTAND), but not of the wild-type strain, indicating a functional link between HMO1 and TAND. Furthermore, Δhmo1 exhibits synthetic growth defects in some spt15 (TBP) and toa1 (TFIIA) mutants while it rescues growth defects of some sua7 (TFIIB) mutants. Importantly, Δhmo1 causes an upstream shift in transcriptional start sites of RPS5, RPS16A, RPL23B, RPL27B and RPL32, but not of RPS31, RPL10, TEF2 and ADH1, indicating that HMO1 may participate in start site selection of a subset of class II genes presumably via its interaction with TFIID.

Yeast HMO1: Linker Histone Reinvented

“…A number of transcription factors have been reported to contribute to the regulation of RP gene activity, including Rap1p, which binds the majority of RP genes and forms nucleosome-free regions in target promoters (155,166). HMO1 binds RP gene promoters with variable occupancy and has been implicated in preinitiation complex (PIC) assembly by covering a nucleosome-free region and in the recruitment of the transcription factor Fhl1p (forkhead like) (149,150,155,167,168).…”

“…The TAF1 N-terminal domain (TAND) inhibits the binding of TBP to the TATA element (169); it has been reported that HMO1 interacts with TBP and TAND and that HMO1 deletion decreases the transcription of TAND-dependent genes, suggesting that HMO1 prevents inhibitory TBP-TAND interactions. In addition, an interaction between HMO1 and TFIID was suggested by the observation that HMO1 deletion causes an upstream shift in transcription start sites of genes under the control of HMO1-enriched promoters but not of genes under the control of promoters with limited HMO1 occupancy (168). This shift in the transcriptional start site was subsequently linked to the ability of HMO1 to mask a nucleosome-free region to prevent inappropriate PIC assembly (167).…”

Both HMG boxes in Hmo1 are essential for DNA binding in vitro and in vivo

“…TSSs were analyzed by primer extension analysis as described previously. 19) The TK3212 (endogenous RPS5) and KK288 (RPS5*p+HIS3 reporter) primers were used for the analysis. Electrophoretic images were acquired by exposing gels to imaging plates, and were analyzed by image analyzer BAS2500 (Fuji Film).…”

“…15) Immunoprecipitation was performed using Dynabeads Protein G (Invitrogen) and an anti-FLAG monoclonal antibody (Sigma-Aldrich; M2) or anti-Hmo1 polyclonal antibody. 19) Real-time quantitative PCR analyses were performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems) and the Mx3000P qPCR System (Agilent Technologies). Each experiment was performed in triplicate and the mean and standard deviations of the ratio of immunoprecipitated DNA to input DNA (IP/input) were calculated.…”

“…Hmo1 regulates the transcription of RPGs by facilitating the binding of Fhl1/ Ifhl1 and TFIID to the promoter and by masking a wide nucleosome-free region between the upstream activating sequence (UAS) and core promoter to direct the assembly of the preinitiation complex (PIC) to a biologically relevant site. 15,16,[18][19][20] Although recent studies have revealed the roles of Hmo1 at RPG promoters, the mechanisms by which Hmo1 binds specifically to these promoters remain unclear.…”