CHARACTERIZATION OF THE PROMOTER REGION OF THE RAT TESTIS-SPECIFIC HISTONE H1T GENE

Histone H1t is synthesized only in male germ cells during the tate pac hytene stage of meiosis and is retained in spermatids until the nucleu s elongates. Transgenic experiments suggest that spermatocyte-directin g sequences lie within 140 base pairs of the cap site. To study the me chanism of this specificity we compared the DNase I footprints made on the immediate promoter regions of H1t and H1d (a typical somatic H1) by testis and liver extracts and observed both common and differential ly protected regions. The common footprints of H1t included an Sp1 con sensus (GC box 1) and a CCAAT motif. Electrophoretic mobility shift as says (EMSA) identified ubiquitous binding factors for GC box 1 and a b inding factor for the CCAAT element that we identified immunologically as H1TF2. H1t-specific footprints occurred over the palindrome CCTAGG and a GC-rich sequence downstream of the TATA box (GC box 2). EMSA an alysis of the palindrome identified testis-specific as well as ubiquit ous binding factors. UV irradiation of a palindrome-binding reaction g enerated a cross-linked doublet of about 50 kDa from both testis and l iver. Protein factors that bound to the GC box 2 sequence were similar from testis and liver, and GC box 1 and an Sp1 consensus competed for them. In vitro transcription directed by H1t occurred at comparable l evels in testis and liver extracts. The importance of both GC box 1 an d CCAAT elements was demonstrated by deletion analysis and by oligonuc leotide competition. No dependence on the H1t palindrome was observed for in vitro transcription.