More Than 1,001 Problems with Protein Domain Databases: Transmembrane Regions, Signal Peptides and the Issue of Sequence Homology

Large-scale genome sequencing gained general importance for life science because functional annotation of otherwise experimentally uncharacterized sequences is made possible by the theory of biomolecular sequence homology. Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. Having the same fold imposes strict conditions over the packing in the hydrophobic core requiring similarity of hydrophobic patterns. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Thus, matching of SPs/TMs creates the illusion of matching hydrophobic cores. Therefore, inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment-mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, we show explicit examples that the scores of clearly false-positive hits, even in global-mode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, we find that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, false-positive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. We suggest a workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users

Molecular cloning, chromosomal mapping, and developmental expression of a novel protein tyrosine phosphatase-like gene.

Protein tyrosine phosphatases (PTPs) mediate the dephosphorylation of phosphotyrosine. PTPs are known to be involved in many signal transduction pathways leading to cell growth, differentiation, and oncogenic transformation. We have cloned a new family of novel protein tyrosine phosphatase-like genes, the Ptpl (protein tyrosine phosphatase-like; proline instead of catalytic arginine) gene family. This gene family is composed of at least three members, and we describe here the developmental expression pattern and chromosomal location for one of these genes, Ptpla. In situ hybridization studies revealed that Ptpla expression was first detected at embryonic day 8.5 in muscle progenitors and later in differentiated muscle types: in the developing heart, throughout the liver and lungs, and in a number of neural crest derivatives including the dorsal root and trigeminal ganglia. Postnatally Ptpla was expressed in a number of adult tissues including cardiac and skeletal muscle, liver, testis, and kidney. The early expression pattern of this gene and its persistent expression in adult tissues suggest that it may have an important role in the development, differentiation, and maintenance of a number of different tissue types. The human homologue of Ptpla (PTPLA) was cloned and shown to map to 10p13-p14

SOX2 Functions to Maintain Neural Progenitor Identity

AbstractNeural progenitors of the vertebrate CNS are defined by generic cellular characteristics, including their pseudoepithelial morphology and their ability to divide and differentiate. SOXB1 transcription factors, including the three closely related genes Sox1, Sox2, and Sox3, universally mark neural progenitor and stem cells throughout the vertebrate CNS. We show here that constitutive expression of SOX2 inhibits neuronal differentiation and results in the maintenance of progenitor characteristics. Conversely, inhibition of SOX2 signaling results in the delamination of neural progenitor cells from the ventricular zone and exit from cell cycle, which is associated with a loss of progenitor markers and the onset of early neuronal differentiation markers. The phenotype elicited by inhibition of SOX2 signaling can be rescued by coexpression of SOX1, providing evidence for redundant SOXB1 function in CNS progenitors. Taken together, these data indicate that SOXB1 signaling is both necessary and sufficient to maintain panneural properties of neural progenitor cells

SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse

Two lines of evidence suggest that the Sry-related gene Sox9 is important for chondrogenesis in mammalian embryos. Sox9 mRNA is expressed in chondrogenic condensations in mice, and mutations in human SOX9 are known to cause skeletal dysplasia. We show here that mouse SOX9 protein is able to bind to a SOX/SRY consensus motif in DNA and contains a modular transcriptional activation domain, consistent with a role for SOX9 as a transcription factor acting on genes involved in cartilage development. One such gene is Col2a1, which encodes type II collagen, the major structural component of cartilage. We have compared, in detail, the expression of Sox9 and Col2a1 during mouse development. In chondrogenic tissues the expression profiles of the two genes were remarkably similar. Coexpression was detected in some nonchondrogenic tissues such as the notochord, otic vesicle, and neural tube, but others such as heart and lung differed in their expression of the two genes. Immunohistochemistry using an antibody specific for SOX9 revealed that expression of SOX9 protein mirrored the distribution of Sox9 mRNA. Our results suggest that SOX9 protein is involved in the regulation of Col2a1 during chondrogenesis, but that this regulation is likely to depend on additional cofactors

Role of an ER stress response element in regulating the bidirectional promoter of the mouse <it>CRELD2 </it>- <it>ALG12 </it>gene pair

<p>Abstract</p> <p>Background</p> <p>Recently, we identified <it>cysteine-rich with EGF-like domains 2 </it>(<it>CRELD2</it>) as a novel endoplasmic reticulum (ER) stress-inducible gene and characterized its transcriptional regulation by ATF6 under ER stress conditions. Interestingly, the <it>CRELD2 </it>and <it>asparagine-linked glycosylation 12 homolog </it>(<it>ALG12</it>) genes are arranged as a bidirectional (head-to-head) gene pair and are separated by less than 400 bp. In this study, we characterized the transcriptional regulation of the mouse <it>CRELD2 </it>and <it>ALG12 </it>genes that is mediated by a common bidirectional promoter.</p> <p>Results</p> <p>This short intergenic region contains an ER stress response element (ERSE) sequence and is well conserved among the human, rat and mouse genomes. Microarray analysis revealed that <it>CRELD2 </it>and <it>ALG12 </it>mRNAs were induced in Neuro2a cells by treatment with thapsigargin (Tg), an ER stress inducer, in a time-dependent manner. Other ER stress inducers, tunicamycin and brefeldin A, also increased the expression of these two mRNAs in Neuro2a cells. We then tested for the possible involvement of the ERSE motif and other regulatory sites of the intergenic region in the transcriptional regulation of the mouse <it>CRELD2 </it>and <it>ALG12 </it>genes by using variants of the bidirectional reporter construct. With regards to the promoter activities of the <it>CRELD2</it>-<it>ALG12 </it>gene pair, the entire intergenic region hardly responded to Tg, whereas the <it>CRELD2 </it>promoter constructs of the proximal region containing the ERSE motif showed a marked responsiveness to Tg. The same ERSE motif of <it>ALG12 </it>gene in the opposite direction was less responsive to Tg. The direction and the distance of this motif from each transcriptional start site, however, has no impact on the responsiveness of either gene to Tg treatment. Additionally, we found three putative sequences in the intergenic region that antagonize the ERSE-mediated transcriptional activation.</p> <p>Conclusions</p> <p>These results show that the mouse <it>CRELD2 </it>and <it>ALG12 </it>genes are arranged as a unique bidirectional gene pair and that they may be regulated by the combined interactions between ATF6 and multiple other transcriptional factors. Our studies provide new insights into the complex transcriptional regulation of bidirectional gene pairs under pathophysiological conditions.</p

Combinatorial regulation of optic cup progenitor cell fate by SOX2 and PAX6

In humans, haploinsufficiency of either SOX2 or PAX6 is associated with microphthalmia, anophthalmia or aniridia. In this study, through the genetic spatiotemporal specific ablation of SOX2 on both wild-type and Pax6-haploinsufficent backgrounds in the mouse, we have uncovered a transcriptionally distinct and developmentally transient stage of eye development. We show that genetic ablation of SOX2 in the optic cup results in complete loss of neural competence and eventual cell fate conversion to non-neurogenic ciliary epithelium. This cell fate conversion is associated with a striking increase in PAX6, and genetically ablating SOX2 on a Pax6-haploinsufficient background partially rescues the Sox2-mutant phenotype. Collectively, these results demonstrate that precise regulation of the ratio of SOX2 to PAX6 is necessary to ensure accurate progenitor cell specification, and place SOX2 as a decisive factor of neural competence in the retina