Comparative sequence and x-inactivation analyses of a domain of escape in human xp11.2 and the conserved segment in mouse

Abstract

We have performed X-inactivation and sequence analyses on 350 kb of sequence from human Xp11.2, a region shown previously to contain a cluster of genes that escape X inactivation, and we compared this region with the region of conserved synteny in mouse. We identified several new transcripts from this region in human and in mouse, which defined the full extent of the domain escaping X inactivation in both species. In human, escape from X inactivation involves an uninterrupted 235-kb domain of multiple genes. Despite highly conserved gene content and order between the two species, Smcx is the only mouse gene from the conserved segment that escapes inactivation. As repetitive sequences are believed to facilitate spreading of X inactivation along the chromosome, we compared the repetitive sequence composition of this region between the two species. We found that long terminal repeats (LTRs) were decreased in the human domain of escape, but not in the majority of the conserved mouse region adjacent to Smcx in which genes were subject to X inactivation, suggesting that these repeats might be excluded from escape domains to prevent spreading of silencing. Our findings indicate that genomic context, as well as gene-specific regulatory elements, interact to determine expression of a gene from the inactive X-chromosome.

Figure 1

Schematic diagram of the human domain of escape from Xp11.2 and the corresponding region of conserved synteny in the mouse. Loci that escape are represented by green arrows or lines and those that are inactivated are in red. The X-inactivation status of the human XM_159437-like locus could not be determined (see Results). When known, the transcriptional orientation is indicated by the direction of the arrow. Orthologs are indicated by shaded regions. Sequences designated with an asterisk may not represent independent transcripts (see Results). Alternative names for transcripts/ESTs are as follows: SE20–4/Se20–4 = DXHXS1008E = TSPX/Tspx; AK055575 = SMC1L1 = SB1.8; ADS13 = FLJ32783; W08639 = NM_025660; ADS9 = HADH2.

Figure 2

X-inactivation analyses of representative mouse transcripts. (A) Inactivation status of Kiaa0522 was determined by digesting RT-PCR products with BlpI to distinguish T16H from CAST alleles. T16H and CAST are parental controls. + and – represent RT-PCR reactions performed with and without reverse transcriptase. Only the T16H (active X) allele is expressed in T16H × CAST F₁ females, demonstrating inactivation of the CAST allele of Kiaa0522. DNA from the F₁ females was also amplified and digested to verify the presence and equal amplification of both alleles. Results from two different F₁ females are shown. (B) Inactivation status of AK013346 was determined by sequence analysis of RT-PCR products to distinguish the T16H and CAST alleles, which differed by an A/T polymorphism. DNA sequence from a T16H × CAST F₁ female confirms the presence of both alleles, whereas cDNA sequence from the same female reveals expression of only the T16H (active X) allele, consistent with inactivation of AK013346.

Figure 2

X-inactivation analyses of representative mouse transcripts. (A) Inactivation status of Kiaa0522 was determined by digesting RT-PCR products with BlpI to distinguish T16H from CAST alleles. T16H and CAST are parental controls. + and – represent RT-PCR reactions performed with and without reverse transcriptase. Only the T16H (active X) allele is expressed in T16H × CAST F₁ females, demonstrating inactivation of the CAST allele of Kiaa0522. DNA from the F₁ females was also amplified and digested to verify the presence and equal amplification of both alleles. Results from two different F₁ females are shown. (B) Inactivation status of AK013346 was determined by sequence analysis of RT-PCR products to distinguish the T16H and CAST alleles, which differed by an A/T polymorphism. DNA sequence from a T16H × CAST F₁ female confirms the presence of both alleles, whereas cDNA sequence from the same female reveals expression of only the T16H (active X) allele, consistent with inactivation of AK013346.

Figure 3

X-inactivation analyses of representative human transcripts (KIAA0522, AJ271378, BF364272) in human × rodent somatic cell hybrids. Xi and Xa designate somatic cell hybrids containing an inactive or an active human X-chromosome on a Chinese hamster background, respectively. + and – indicate reactions performed with or without reverse transcriptase. The CHO (Chinese hamster ovary cell line) control shows that primers are specific for human sequence, and the human cDNA (HUM) serves as a positive control. RBMX is a control gene previously shown to be subject to X inactivation (Lingenfelter et al. 2001).

Figure 4

Dot plot of human versus mouse genomic sequence across the human Xp11.2 escape domain, generated using Pip analysis software (http://bio.cse.psu.edu/pipmaker). Human genes, ESTs, and direction of transcription are shown above the plot.

Figure 5

Repetitive DNA content in the domain of escape. (A) Number of repetitive elements in the SMCX/Smcx gene region, the remainder of the human escape domain (between SMCX and HADH2, these two genes being excluded) and the equivalent mouse region, the whole X-chromosome, and the entire genome (see Table 2). Repeats are displayed as number normalized per megabase of DNA. (B) Sliding-window analysis of the distribution of repeats across the region of escape (shaded) and flanking inactivated regions.