Transcription factor RFX4 binding to the testis-specific histone H1t promoter in spermatocytes may be important for regulation of H1t gene transcription during spermatogenesis.

The X-box binding protein RFX4 is highly expressed in testis in contrast with other tissues, but its function there is unknown. Another family member abundant in testis, RFX2, has been shown to bind to the X-Box elements in the promoter of the testis specific histone H1t, which is expressed only in pachytene spermatocytes. RFX proteins are known to dimerize, and there is the possibility that the abundant testis RFX4, which is also expressed in pachytene spermatocytes as shown by RT-PCR and Western blotting, may interact with RFX2 in these cells. In EMSA anti-RFX2 polyclonal antibodies produce a supershifted complex with testis extracts and an X-Box probe. On the other hand, RFX4 polyclonal antibodies do not supershift the complex but appear to enhance formation of the complex. RFX4 appears to co-precipitate with RFX2 in immunoprecipitation, and to co-purify with RFX2 in an affinity purification using a biotinylated X-box affinity probe. In ChIP assays RFX4 also binds to the H1t promoter in vivo. These data suggest a possible regulatory role for RFX4 in transcription of the histone H1t gene during spermatogenesis.

Binding of RFX2 and NF-Y to the testis-specific histone H1t promoter may be required for transcriptional activation in primary spermatocytes.

The testis-specific linker histone H1t is transcribed exclusively in pachytene spermatocytes during spermatogenesis. The H1t promoter contains two imperfect inverted repeats which together comprise the X-box motif that is known to bind the transcription factor regulatory factor X (RFX). Out of all the histone H1 family promoters this motif appears only in the H1t promoter and may contribute to H1t tissue-specific expression. We show by Western blotting, EMSA, ChIP analysis, and real-time RT-PCR that the rat H1t X-box is bound by RFX2 in vivo in spermatocytes. We demonstrate that transcription factor NF-Y binds to the CCAAT-box motif that is located downstream and adjacent to the X-box and that testis NF-Y interacts either directly or indirectly with RFX2. Furthermore, we show that both the X-box and CCAAT-box are required for promoter activity and that co-expression of RFX2 greatly enhances testis histone H1t promoter activity in the GC-1spg germinal cell line.

Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.