Characterization of Xpr (Xpct) reveals instability but no effects on X-chromosome pairing or Xist expression.

X-chromosome inactivation balances X-chromosome dosages in male and female mammals by transcriptionally repressing one X in the female sex. Proper counting and the mutually exclusive choice of active X and inactive X have been hypothesized to involve X-chromosome crosstalk via homologous chromosome pairing. Transient pairing of two female Xs requires noncoding Tsix and Xite. A recent study suggested a new pairing element (Xpr), located ~200 kb upstream of Xist, in the Xpct region. Xpr is proposed to induce pairing and activate Xist expression. Here, we further characterize Xpr and find that the Xpr sequence is unstable when introduced as transgenes into male ES cells. Xpr transgenes show an unusual tendency to disperse throughout the nucleus. However, we observe neither pairing between Xpr alleles nor ectopic Xist expression. In the absence of Tsix, Xpr does not induce inter-allelic Xic interactions. Female ES cells carrying Xpr transgenes are more stable. Nonetheless, pairing also does not seem to occur in female cells. We conclude that, while Xpr contains unusual properties, it most likely does not serve as a pairing or counting element. Differences in statistical methods and controls may explain some of the discrepancies.

A highly distinctive mechanical property found in the majority of human promoters and its transcriptional relevance.

A recent study revealed that TATA boxes and initiator sequences have a common anomalous mechanical property, i.e. they comprise distinctive flexible and rigid sequences when compared with the other parts of the promoter region. In the present study, using the flexibility parameters from two different models, we calculated the average flexibility profiles of 1004 human promoters that do not contain canonical promoter elements, such as a TATA box, initiator (Inr) sequence, downstream promoter element or a GC box, and those of 382 human promoters that contain the GC box only. Here, we show that they have a common characteristic mechanical property that is strikingly similar to those of the TATA box-containing or Inr-containing promoters. Their most interesting feature is that the TATA- or Inr-corresponding region lies in the several nucleotides around the transcription start site. We have also found that a dinucleotide step from -1 to +1 (transcription start site) has a slight tendency to adopt CA that is known to be flexible. We also demonstrate that certain synthetic DNA fragments designed to mimic the average mechanical property of these 1386 promoters can drive transcription. This distinctive mechanical property may be the hallmark of a promoter.

Core promoter elements of eukaryotic genes have a highly distinctive mechanical property.

In spite of the abundant data on DNA sequence, the mechanical aspects of promoter DNA remain poorly understood. We classified 1871 human and 196 mouse RNA polymerase II promoters and investigated average flexibility profiles of the human promoters containing either a TATA box or an initiator (Inr) sequence only. Here, we show that TATA boxes and Inr sequences have a common anomalous mechanical property: they are comprised of distinctively flexible and rigid sequences, compared with the other parts of the promoter region. The +2 position in the Inr consensus sequence does not favor adenine to keep the high flexibility and thus this position is more accurately represented as 'T, G, C>>A'. Additionally, it was also found that DNA region upstream of TATA box or Inr sequence is more rigid than region downstream of each element. These properties may function as a marker for recognition by TATA-binding protein and Inr-binding protein.

Left-handedly curved DNA regulates accessibility to cis-DNA elements in chromatin.

There is little information on chromatin structure that allows access of trans-acting transcription factors. Logically, the target DNA elements become accessible by either exposing themselves towards the environment on the surface of the nucleosome, or making the regulatory region free of the nucleosome. Here, we demonstrate that curved DNA that mimics a negative supercoil can play both roles in the promoter region. By constructing 35 reporter plasmids and using in vivo assay systems, we scrutinized the relationships between upstream DNA geometry, nucleosome positioning and promoter activity. When the left-handedly curved DNA was linked to the herpes simplex virus thymidine kinase (HSV tk) promoter at a specific rotational phase and distance, the curved DNA attracted the nucleosome and the TATA box was thereby left in the linker DNA with its minor groove facing outwards, which led to the activation of transcription. Neither planar curving, nor right-handedly curved DNA nor straight DNA had this effect. Our results seem to provide a clue for solving the problem of why curved DNA is often located near transcriptional control regions.