Histone H1t Gene Research Articles

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.

Transcription factor RFX2 is abundant in rat testis and enriched in nuclei of primary spermatocytes where it appears to be required for transcription of the testis‐specific histone H1t gene

Previous work in our laboratory revealed upregulated transcription of the testis-specific linker histone H1t gene in pachytene primary spermatocytes during spermatogenesis. Using the H1t X-box as an affinity chromatography probe, we identified Regulatory Factor X2 (RFX2), a member of the RFX family of transcription factors, as a nuclear protein that binds the probe. We also showed that RFX2 activated the H1t promoter in transient expression assays. However, other RFX family members have the same DNA-binding domain and they also may regulate H1t gene expression. Therefore, in this study we examined the distribution of RFX2 and other RFX family members in rat testis germinal cells and in several tissues. Among tissues examined, RFX2 is most abundant in testis. Testis RFX2 is most abundant in spermatocytes where transcription of the H1t gene is upregulated and the steady-state H1t mRNA level is high. RFX2 levels decrease but RFX1 levels increase in early spermatids where H1t gene transcription is downregulated. Antibodies against RFX2 generate a shifted band in electrophoretic mobility shift assays (EMSA) using H1t or testisin X-box DNA probes with nuclear proteins from spermatocytes. These data support the hypothesis that RFX2 expression is upregulated in spermatocytes where it participates in activating transcription of the H1t gene and other testis genes. These data also support the possibility that other RFX family members may bind to the H1t promoter in other testis germinal cell types and in nongerminal cells to downregulate H1t gene transcription.

Transcriptional activation of the testis-specific histoneH1t gene by RFX2 may require both proximal promoter X-box elements

The rat testis-specific linker histone H1t gene is transcribed in pachytene primary spermatocytes during spermatogenesis. Our previous work using transgenic mice demonstrated that spermatocyte-specific transcription of the H1t gene is dependent upon a proximal promoter element designated the TE element. TE is composed of two adjacent and inverted imperfect repeat sequences designated TE1 and TE2 and both of these palindromic elements are similar in sequence to the X-box, a DNA consensus sequence that binds regulatory factor X (RFX). RFX2 is the major enriched protein derived from rat testis nuclear extracts when using the TE1 element as an affinity chromatography probe. Co-expression of RFX2 together with an H1t promoted reporter vector in transient expression assays activates the H1t promoter in the GC-2spd germinal cell line, and mutation of either X-box significantly represses activity. However, RFX2 partially reactivates the promoter when either of the X-box elements is independently mutated. In order to totally block reactivation by RFX2, it is necessary to mutate both X-boxes simultaneously. Therefore, RFX2 appears to be able to bind to either X-box independently to partially activate the promoter of the testis-specific histone H1t gene, but simultaneous binding of RFX2 to both X-box elements may be required for maximal promoter activation.

Regulatory factor X2 (RFX2) binds to the H1t/TE1 promoter element and activates transcription of the testis-specific histone H1t gene.

Transcription of the mammalian testis-specific linker histone H1t gene occurs only in pachytene primary spermatocytes during spermatogenesis. Studies of the wild type (Wt) and mutant H1t promoters in transgenic mice show that transcription of the H1t gene is dependent upon the TE promoter element. We purified an 85 kDa protein from rat testis nuclear extracts using the TE1 subelement as an affinity chromatography probe and analysis revealed that the protein was RFX2. The TE1 element is essentially an X-box DNA consensus element and regulatory factor X (RFX) binds specifically to this element. Polyclonal antibodies directed against RFX2 supershift the low mobility testis nuclear protein complex formed in electrophoretic mobility shift assays (EMSA). RFX2 derived from primary spermatocytes, where the transcription factor is relatively abundant, binds with high affinity to the TE1 element. Coexpression of RFX2 together with an H1t promoter/reporter vector activates the H1t promoter in a cultured GC-2spd germinal cell line, but mutation of either the TE1 subelement or the TE2 subelements represses activity. These observations lead us to conclude that the TE1 and TE2 subelements of the testis-specific histone H1t promoter are targets of the transcription factor RFX2 and that this factor plays a key role in activating transcription of the H1t gene in primary spermatocytes. Published 2003 Wiley-Liss, Inc.

Transcriptional repression of the testis-specific histoneH1t gene mediated by an element upstream of the H1/AC box

The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes and may be important for chromatin structure, transcription, and DNA repair during this stage of spermatogenesis. Transcriptional repression of the gene in other cell types is mediated in part by specific proximal and distal promoter elements and in some cell types by methylation of CpG dinucleotides within the promoter. Our laboratory identified a distal promoter element located between 948 and 780 bp upstream from the transcription initiation site and another laboratory identified a GC-rich region between the TATA box and transcription initiation site that contribute to repression. In this article we address transcriptional repression of the histone H1t gene by an element within the proximal promoter. We report discovery of an element designated H1t promoter repressor element (RE) located between −130 and −106 bp that contributes to repression. The findings support the hypothesis that multiple mechanisms are involved in transcriptional repression of the H1t gene. Transcriptional repression mediated by the RE element in NIH 3T3 cells appears to differ significantly from the mechanism mediated by the GC-rich region. Furthermore, binding proteins that form the RE complex are not present in rat testis where the gene is actively transcribed. Our findings provide a molecular basis for histone H1t gene repression.

Specific binding of nuclear proteins to a bifunctional promoter element upstream of the H1/AC box of the testis-specific histone H1t gene.

The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes during spermatogenesis. Studies with transgenic mice show that 141 base pairs (bp) of the H1t proximal promoter accompanied with 800 bp of downstream sequence are sufficient for tissue-specific transcription. Nuclear proteins from testis and pachytene spermatocytes produce footprints spanning the region covering the repressor element (RE) from 100 to 125 nucleotides upstream of the H1t transcriptional initiation site. Only testis nuclear proteins bind to the 5'-end of the element and produce a unique, low-mobility complex in electrophoretic mobility shift assays. This testis complex is distinct from the complex formed by a repressor protein derived from several cell lines that binds to the 3'-end of the element. The testis complex band is formed when using nuclear proteins from primary spermatocytes, where the H1t gene is transcribed, and band intensity drops 70%-80% when using nuclear proteins from early spermatids, where H1t gene transcription ceases. Protein-DNA cross-linking experiments using testis nuclear proteins produce electrophoretic bands of 59, 52, and 50 kDa on SDS/PAGE gels.

H1t/GC-box and H1t/TE1 element are essential for promoter activity of the testis-specific histone H1t gene.

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue-specific expression of the gene is mediated primarily through elements located within the proximal promoter. Previous work in transgenic animals identified a unique 40-base pair promoter element designated H1t/TE that is essential for spermatocyte-specific expression. The H1t/TE element contains three subelements designated TE2, GC-box, and TE1 based on in vitro footprinting and electrophoretic mobility shift assays. Because GC-box is a consensus site for binding of Sp transcription-factor family members, experiments were performed demonstrating that two Sp family members, Sp1 and Sp3, were present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids. A 95- to 105-kDa form of Sp1 is most abundant in the tissues and cell lines examined, but a 60-kDa form of Sp1 is the most abundant species in spermatocytes and early spermatids. Further examination of Sp1 and Sp3 from adult testis, primary spermatocytes, and early spermatids showed that they can bind to the H1t/TE element. In order to determine the contributions of the subelements to H1t transcription, we mutated each of them in H1t promoter luciferase reporter vectors. Mutation of the GC-box and TE1 subelement reduced expression 77% and 49%, respectively, compared with the wild-type H1t promoter in transient expression assays in a testis GC-2spd cell line that was derived from germinal cells. These studies suggest that Sp transcription factors may be involved in transcription of the H1t gene and the GC-box and the TE1 subelement are required for activation of the H1t promoter.

Sp1 and Sp3 activate the testis-specific histone H1t promoter through the H1t/GC-box.

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in large part through elements located within the proximal promoter. Previous work in transgenic animals showed that a unique 40 bp promoter element designated H1t/TE is essential for spermatocyte-specific expression. The H1t/TE element contains a GC-box, which is a perfect consensus binding site for members of the Sp family of transcription factors. We have shown that Sp1 and Sp3 are present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids and that they can bind to the H1t/GC-box. Mutagenesis of the GC-box reduced H1t promoter activity. Furthermore, a CpG dinucleotide within the GC-box was totally unmethylated in rat testis primary spermatocytes where the gene is transcribed but it was methylated in liver where the gene is silenced. These previous studies supported the importance of the GC-box and suggested that Sp transcription factors contribute to expression of the H1t gene. In this study, we show that co-transfection of Sp1 and Sp3 expression vectors leads to an upregulation of histone H1t promoter activity in several cell lines including testis GC-2spd cells. However, very low H1t promoter activity is seen in GC-2spd cells grown at 39 degrees C, which correlates with lower levels of Sp1 and Sp3 in these cells grown at this elevated temperature. Upregulation of the H1t promoter by Sp1 and Sp3 was also seen in cotransfected NIH3T3 and C127I cell lines. On the other hand, co-transfection of the Sp1 and Sp3 expression vectors does not lead to upregulation of activity of the cell-cycle dependent histone H1d promoter.

Testis-specific histone H1t gene is hypermethylated in nongerminal cells in the mouse.

The testis-specific histone H1t gene is expressed only in pachytene primary spermatocytes during spermatogenesis. There is a correlation between the specific binding of testis nuclear proteins to a rat histone H1t promoter sequence, designated the H1t/TE element, and the onset of transcription in primary spermatocytes. Our laboratory has shown that mice bearing the rat gene with a deletion of the TE promoter element and replacement with a heterologous stuffer DNA fragment fail to express the rat H1t transgene in any tissue. In this study we report that five CpGs located within the H1t proximal promoter, including two CpGs located within the essential TE promoter element, contain unmethylated cytosines in vivo in genomic DNA derived from primary spermatocytes where the H1t gene is expressed. All seven CpGs are hypermethylated in vivo in genomic DNA derived from liver cells where gene expression is repressed. Further, in vitro methylation of an H1t promoter-driven reporter plasmid markedly reduced expression in a transient transfection assay system. These results suggest that cytosine methylation may contribute to the transcriptional silencing of the testis-specific histone H1t gene in nonexpressing tissues such as liver.

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Binding of nuclear proteins to an upstream element involved in transcriptional regulation of the testis-specific histone H1t gene.

The testis-specific histone H1t is synthesized during spermatogenesis exclusively in late pachytene primary spermatocytes. Transcription of the H1t gene is repressed in every tissue except testis. Within the testis, transcription is repressed during development before the spermatocyte stage and in later stages of germinal cell maturation. Mechanisms involved in transcriptional repression of the H1t gene are unknown. To assess the contribution of upstream H1t promoter sequence to transcriptional silencing in nonexpressing cells, H1t-promoted reporter vectors were constructed using pGL3 Basic. Transient expression assays with these reporter vectors driven by H1t promoter deletions allowed us to identify a region from 948 to 780 bp upstream from the H1t transcriptional initiation site that functions as a silencer. Examination of nuclear protein binding to this DNA regulatory region by electrophoretic mobility shift assays using extracts from C127I cells, rat testis, and pachytene spermatocytes revealed a low mobility band produced only by nuclear proteins derived from nonexpressing cells that may contain proteins that repress H1t gene transcription.

Localization of upstream elements involved in transcriptional regulation of the rat testis-specific histone H1t gene in somatic cells.

The testis-specific histone H1t is synthesized exclusively in late pachytene primary spermatocytes during spermatogenesis. The mechanisms involved in transcriptional repression of the H1t gene during development before the spermatocyte stage and in later stages of germinal cell maturation and in nonexpressing somatic tissues are unknown. To assess the contribution of the upstream DNA sequence to H1t transcriptional silencing in nonexpressing cells, a set of histone H1t-promoted reporter vectors was constructed. Transient transfection of mouse C127I cells with these reporter vectors allowed us to identify a transcriptional silencer located between 948 base pairs (bp) and 780 bp upstream from the H1t transcriptional initiation site. Histone H1t-promoted luciferase activity increased 4-fold when the region between 948 bp and 875 bp upstream from the transcriptional initiation site was eliminated. Addition of a 73-bp rat H1t promoter fragment (-948 to -875, containing the 5' portion of the silencer region) to a site immediately upstream from the histone H1d proximal promoter led to significantly reduced luciferase expression upon transient transfection (56% in C127I cells and 44% in HeLa cells). Nuclear proteins were found to bind to DNA within the H1t silencer region when assayed by in vitro deoxyribonuclease (DNase) I footprinting. Thus, our data suggest that an active transcriptional silencer mechanism involving a specific and autonomous H1t promoter element (nucleotides -948/-875) may be operative to minimize expression of the H1t gene in nontesticular cells.

The TE promoter element of the histone H1t gene is essential for transcription in transgenic mouse primary spermatocytes.

Transcriptional activation of the testis-specific histone H1t gene occurs in pachytene primary spermatocytes during spermatogenesis. Specific binding of testis nuclear proteins to a rat histone H1t promoter sequence, designated the H1t/TE element, correlates with the onset of transcription. This element, located between the H1t/AC box and the H1t/CCAAT box, contains inverted repeats of a shorter element. When the native rat H1t gene along with flanking sequences, including 2453 base pairs (bp) upstream and 3784 bp downstream from the coding region, was microinjected into mouse embryos, the offspring of the resulting transgenic mice transcribed the transgene in a tissue-specific manner and only in primary spermatocytes. In the present study the TE promoter element was deleted and replaced with a heterologous stuffer DNA fragment. When the mutant rat DNA fragment was used to create transgenic mice, offspring of the mice bearing the promoter mutation did not transcribe the rat H1t gene in any tissue. On the other hand, transcription of the rat H4t transgene, which is located approximately 1.5 kilobases downstream from the H1t gene, occurred in these animals. Therefore, these studies support the hypothesis that the TE element is essential for enhanced testis-specific transcription of the H1t gene in primary spermatocytes.

The haemochromatosis candidate gene HFE (HLA-H) of man and mouse is located in syntenic regions within the histone gene cluster.

The HFE (HLA-H) gene is a strong candidate gene for hereditary haemochromatosis and was localized on the short arm of chromosome 6 to 6p21.3-p22. In addition, the sequence of the homologous mouse and rat cDNA and a partial sequence from the mouse gene have been reported recently. In this report, we describe the location of the human and the mouse HFE (HLA-H) gene within the histone gene clusters on the human chromosome 6 and the mouse chromosome 13. Both the human and the murine gene were located on syntenic regions within the histone gene clusters in the vicinity of the histone H1t gene. The genomic sequence of the human HFE (HLA-H) gene and the 3' portion of the homologous mouse gene were determined. Comparison of the genomic sequences from man and mouse and the cDNA sequence from rat shows significant similarities, also beyond the transcribed region of the mouse gene.

The Testis-specific Histone H1t Gene Is Strongly Repressed by a G/C-rich Region Just Downstream of the TATA Box

H1t is a testis-specific histone 1 variant restricted to the male germ line and expressed only in pachytene spermatocytes. Understanding the regulation of the H1t gene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1t is not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1t and a major somatic H1 gene (H1d) by introduction of suitable restriction sites just 5' of the TATA box and 3' of the conserved H1 AC box. Hybrid promoters were joined to a lacZ reporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1d promoter was 20-fold stronger than the H1t promoter. Much of this difference in activity was traced to inhibitory sequences immediately downstream of the TATA box in H1t, although sequences upstream of the H1t AC box and within the H1t 5'-untranslated region played some role as well. A series of deletions and short oligonucleotide mutations scanned across the region between the TATA box and cap site identified two tracts of C (GC box 2) as the inhibitory sequences. While both Sp1 and Sp3 bind to this region weakly in vitro, they are unlikely to be responsible for the inhibitory effect of GC box 2, and additional binding proteins (CTB-4 and CTB-5) were identified by electrophoretic mobility shift assays as better candidates for mediating the repressive effect. When repression of the H1t promoter was relieved by mutation of GC box 2, additional mutations introduced into GC box 1 upstream of the CAAT box led to a large decrease in activity, indicating that these two G/C-rich elements have opposite effects on promoter activity.

Regulation of transcription of the testis-specific histone H1t gene by multiple promoter elements.

This is a review of mechanisms that contribute to testis-specific transcription of the histone H1t gene. The mammalian testis-specific histone H1t gene is transcribed only in primary spermatocytes during spermatogenesis. Linker histones bind to DNA and contribute to chromatin condensation by formation of the 30 nm chromatin fiber. Furthermore, linker histones contribute to regulation of transcription of specific genes. Histone H1t, which binds more weakly to DNA than the other six known linker histones, is expressed in cells that are involved in the essential processes of crossing over and mismatch repair of DNA and in cells that undergo a dramatic alteration in gene expression. However, contributions of this linker histone to these processes are unknown. Subtle differences are found in the H1t promoter compared to the other H1 promoters. Nevertheless, several lines of evidence support the hypothesis that a sequence element designated TE that is located within the H1t promoter is essential for enhanced testis-specific transcription of this gene. Transgenic mice bearing a rat H1t transgene which contains a replacement of the TE element with stuffer DNA fail to express rat H1t mRNA. In addition, an upstream sequence appears to function as a silencer element that leads to transcriptional repression of the H1t gene in nongerminal cells. Thus, multiple promoter elements appear to contribute to regulation of transcription of the histone H1t gene.

Characterization of the promoter region of the rat testis-specific histone H1t gene.

Histone H1t is synthesized only in male germ cells during the late pachytene stage of meiosis and is retained in spermatids until the nucleus elongates. Transgenic experiments suggest that spermatocyte-directing sequences lie within 140 base pairs of the cap site. To study the mechanism 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 differentially protected regions. The common footprints of H1t included an Sp1 consensus (GC box 1) and a CCAAT motif. Electrophoretic mobility shift assays (EMSA) identified ubiquitous binding factors for GC box 1 and a binding 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 analysis of the palindrome identified testis-specific as well as ubiquitous binding factors. UV irradiation of a palindrome-binding reaction generated a cross-linked doublet of about 50 kDa from both testis and liver. 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 levels in testis and liver extracts. The importance of both GC box 1 and CCAAT elements was demonstrated by deletion analysis and by oligonucleotide competition. No dependence on the H1t palindrome was observed for in vitro transcription.

Characterization of the mouse histone gene cluster on chromosome 13: 45 histone genes in three patches spread over 1Mb.

The histone gene cluster on mouse chromosome 13 has been isolated and characterized. Using overlapping YAC clones containing histone genes from chromosome 13, a contig of approximately 2 Mb has been defined. It contains 45 histone genes, organized in three patches containing tightly clustered genes. An 80-kb patch (patch III) containing 12 histone genes is near one end of the contig, and a similar-sized patch (patch I) containing 15 histone genes is near the other end of the contig, located at least 500 kb from the central patch (patch II) of histone genes. The entire cluster contains six histone H1 genes, including the testis-specific histone H1t gene that maps to the middle of the cluster. All nine histone H3 genes in this cluster have been sequenced, and their level of expression determined. Each histone H3 gene is distinct, with five genes encoding the H3.2 protein subtype and four genes encoding the H3.1 protein. They are all expressed, with each histone H3 gene accounting for a small proportion of the total histone H3 mRNA.

Binding of nuclear proteins to a conserved histone H1t promoter element suggests an important role in testis-specific transcription

The testis-specific histone H1t gene is transcribed only in primary spermatocytes during spermatogenesis. Recently, expression of the rat gene was shown to be limited to primary spermatocytes in transgenic mice, revealing that promoter elements sufficient for regulating tissue-specific transcription were present in the cloned rat gene. In this study the mouse histone H1t gene has been cloned, and sequenced and its promoter region has been compared to the rat H1t promoter with regard to conserved elements and protein binding activity. The amino acid sequence of each of the three H1t coding region domains is conserved when compared to the homologous domain in H1t derived from other species. H1t mRNA is found only in testis, where it accumulates to a high steady-state level, and examination of enriched testis cell populations shows that expression is limited to primary spermatocytes. Protein binding assays using nuclear extracts from various mouse tissues reveal testis-specific binding to TE1 and TE2, imperfect inverted repeat elements within the larger TE element. Although the H1t promoter contains an Sp1 consensus motif within the H1t/TE element, binding of testis Sp1 to the motif could not be detected using specific anti-Sp1 antibodies. © 1996 Wiley-Liss, Inc. 1 This article is a US Government work and, as such, is in the public domain in the United States of America.

Binding of nuclear proteins to a conserved histone H1t promoter element suggests an important role in testis-specific transcription.

The testis-specific histone H1t gene is transcribed only in primary spermatocytes during spermatogenesis. Recently, expression of the rat gene was shown to be limited to primary spermatocytes in transgenic mice, revealing that promoter elements sufficient for regulating tissue-specific transcription were present in the cloned rat gene. In this study the mouse histone H1t gene has been cloned, and sequenced and its promoter region has been compared to the rat H1t promoter with regard to conserved elements and protein binding activity. The amino acid sequence of each of the three H1t coding region domains is conserved when compared to the homologous domain in H1t derived from other species. H1t mRNA is found only in testis, where it accumulates to a high steady-state level, and examination of enriched testis cell populations shows that expression is limited to primary spermatocytes. Protein binding assays using nuclear extracts from various mouse tissues reveal testis-specific binding to TE1 and TE2, imperfect inverted repeat elements within the larger TE element. Although the H1t promoter contains an Sp1 consensus motif within the H1t/TE element, binding of testis Sp1 to the motif could not be detected using specific anti-Sp1 antibodies.