The enigma of speech and language acquisition in human - A genetic approach

The use of language is one of the most vital traits that distinguish human from other species. The gene FOXP2 (fork head box P2) is relevant to the human ability to develop language. FOXP2 is situated on human chromosome 7q31, and its major splice form encodes a protein of 715 amino acids. It contains a glutamine rich region consisting of two adjacent polyglutamine tracts, encoded by mixtures of CAG and CAA repeats. Two functional copy of FOXP2 seem to be required for acquisition of normal language in human. The chimpanzee, gorilla and the rhesus macaque FOXP2 proteins are all identical to each other and carry only one difference from the mouse and two differences from the human protein, whereas the orangutan two differences from the mouse and three from humans. This gene is also important on particular aspects of cognition awareness or grammatical processing.

Identification of PU.1 Target Genes That Are Dependent on Specific Functional Domains of the Transcription Factor PU.1.

The expression of PU.1, a member of the ets transcription factor family, is limited to the hematopoietic lineage. Using knockout and reduced PU.1 expression and gain of function mice as a model system, it has been demonstrated that PU.1 plays a key role in early myeloid and lymphoid fate decision and at later stages of myeloid differentiation and function. In PU.1 (Sfpi1) null mice, dendritic cells and monocytes could not be identified, but early myeloid progenitors and immature neutrophils were present. In mice with reduced PU.1 expression monocyte development was hindered, immature neutrophil development ensued, and the mice developed acute myeloid leukemia. The different domains of the PU.1 transcription factors have been attributed to distinct functions in early myeloid fate decision. The PU.1 transcription factor is composed of 3 major domains: transactivation, PEST, and DNA binding. We have used an immature myeloid cell line (503) derived from the Sfpi1-null mouse to analyze the effect of lentiviral gene delivery of PU.1 mutants lacking specific PU.1 domains, on differentiation of 503 cells, and to identify PU.1 target genes that are dependent on these specific domains for their expression. We found that a PU.1 mutant lacking the acidic domain leads to differentiation of the 503 cells towards neutrophil lineage, measured by specific lineage specific surface markers. Expression of a PU.1 mutant lacking the glutamine rich region of the transactivation domain did not promote differentiation to neutrophils. Moreover, the glutamine rich and acidic motifs are not necessary for the expression of the CD11b. Despite the independence of CD11b expression from both transactivation domain motifs, its expression was dependent on PU.1 DNA binding. Using cDNA microarray analysis, we identified PU.1 target genes that are regulated by specific domains of the PU.1 transcription factor. In our Affymetrix cDNA microarray analysis we identified over 1300 gene changes that are exclusively dependent on either the acidic domain or the glutamine rich region of the PU.1 transactivation domain for regulation. In addition, we found 33 PU.1- genes that are repressed by the PU.1 mutant lacking the acidic domain, but activated by the glutamine rich region PU.1 mutant, and 38 genes that are regulated in the opposite direction, thus allowing us to begin to identify the hierarchy of the PU.1-regulated gene cascade assocated with cellular function. Currently, we are investigating the biological significance of some of these differentially expressed target genes. For example, one of the identified genes, Neutrophil Elastase (ELA2), is highly upregulated in the 503 cells expressing full length PU.1, or the PU.1 mutant lacking the acidic domain, but is profoundly downregulated in the 503 cell line expressing the glutamine rich region deletion mutant. It has been shown that ELA2 deficient mice are protected form acute promyelocytic leukemia. We are currently investigating the functional role of the PU.1-dependent regulation of ELA2 in our model system. In addition, we are identifying target genes that are regulated exclusively by other PU.1 domains, i.e. the PEST domain. Taken together, we identified and are analyzing the functional importance of specific target genes in PU.1-dependent myeloid differentiation that are regulated by different functional domains of the PU.1 transcription factor.

The DMT1 Associated Protein (DAP) Regulates Iron Uptake by Erythroid Cells.

The divalent metal transporter 1 (DMT1) is essential for cellular iron uptake both in the intestine and in erythroid cells. We have previously shown that with iron feeding the DMT1 expressed on the brush border membrane (BBM) of the intestine undergoes endocytosis (Am. J. Physiol. 283, G965, 2002). Using the yeast two-hybrid system we have isolated a cDNA clone encoding a protein of 526-amino acid residues with a calculated molecular mass of 60 kDa, which interacts with the C-terminus of DMT1 expressed from the IRE containing mRNA (Blood ,100, 7a, 2002). The ORF of the rat protein has been fully sequenced (Genbank #AY336075) and is now designated DMT1 associated protein (DAP). DAP is ubiquitously expressed and is especially abundant in the rat intestine and colon. In rat duodenum DAP is colocalized with DMT1 in the BBM. Both by salt and pH elution DAP was demonstrated to be a peripheral membrane protein. With iron feeding both DMT1 and DAP translocate: DAP moves from the BBM to basolateral membrane (BLM) with DMT1 and some of DMT1 undergoes endocytosis and is found in cytopasmic vesicles. Immunoprecipitation and pull-down assays confirm the interaction of DAP and DMT1 in the BBM vesicles (MMBV). We have analyzed the function of DAP by exploring the role of various consensus sequences in the DAP ORF in the cellular localization of the protein. By sequence motif analysis DAP has a nuclear localization signal, Glutamic acid-rich region, Glutamine-rich region, Arginine-rich region, PKC phosphorylation sites and GOLD domain (Golgi dynamics). The region of DAP protein interacting with the COOH-terminal cytoplasmic domain of DMT1(IRE) was found to be from 171aa to 331aa which contains Glutamic acid-rich region, Glutamine-rich region and Arginine-rich region. Immunocytochemistry confirmed that DAP is localized in the nuclei and the Golgi complex of K562, MDCK, Hela, Cos1 cells, and Caco2 (where DAP is found also in BBM). GFP-fusion constructs containing the DAP nuclear localization signal (amino acids 171–277) or GOLD domain (amino acid 278–526) were transfected into COS-1 and K562 cells and specificity of intracellular localization confirmed by fluorescence confocal microscopy. DAP expression was controlled by cellular iron content: Cells which were iron depleted had increased levels of DAP protein while cells which were iron replete had decreased DAP protein. The regulation by iron was post-transcriptional as iron levels had no affect on DAP mRNA. The levels of DAP expression was also seen to affect iron uptake. Over expression of the region of DAP which binds to DMT1 by transfection of the appropriate construct into K562 cells decreased iron uptake as measured by an increase of transferrin receptor expression and decreased levels of ferritin. Elevated DAP had no affect on endogenous DMT1 expression. Conversely, when siRNAs were used to decrease DAP mRNA in K562 cells there was increased iron uptake with decreased expression of transferrin receptor and increased ferritin expression. In these experiments siRNAs reduced DAP expression by about 60%. This is the first demonstration that a protein which interacts with DMT1 can regulate the uptake of iron into the cell and suggests that DAP may act in a regulatory pathway for iron homeostasis.

Characterization of the Two Coactivator-interacting Surfaces of the Androgen Receptor and Their Relative Role in Transcriptional Control*

The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer.

"In vivo" toxicity of a truncated version of the Drosophila Rst-IrreC protein is dependent on the presence of a glutamine-rich region in its intracellular domain

The roughest-irregular chiasm C ( rst-irreC) gene of Drosophila melanogaster encodes a transmembrane glycoprotein containing five immunoglobulin-like domains in its extracellular portion and an intracytoplasmic tail rich in serine and threonine as well some conserved motifs suggesting signal transduction activity. In the compound eye, loss-of-function rst-irreC mutants lack the characteristic wave of programmed cell death happening in early pupa and which is essential for the elimination of the surplus interommatidial cells. Here we report an investigation on the role played by the Rst-irreC molecule in triggering programmed cell death. "In vivo" transient expression assays showed that deletion of the last 80 amino acids of the carboxyl terminus produces a form of the protein that is highly toxic to larvae. This toxicity is suppressed if an additional 47 amino acid long, glutamine-rich region ("opa-like domain"), is also removed from the protein. The results suggest the possibility that the opa-like domain and the carboxyl terminus act in concert to modulate rst-irreC function in apoptosis, and we discuss this implication in the context of the general mechanisms causing glutamine-rich neurodegenerative diseases in humans.

The AF1 and AF2 Domains of the Androgen Receptor Interact with Distinct Regions of SRC1

ABSTRACT The androgen receptor is unusual among nuclear receptors in that most, if not all, of its activity is mediated via the constitutive activation function in the N terminus. Here we demonstrate that p160 coactivators such as SRC1 (steroid receptor coactivator 1) interact directly with the N terminus in a ligand-independent manner via a conserved glutamine-rich region between residues 1053 and 1123. Although SRC1 is capable of interacting with the ligand-binding domain by means of LXXLL motifs, this interaction is not essential since an SRC1 mutant with no functional LXXLL motifs retains its ability to potentiate androgen receptor activity. In contrast, mutants lacking the glutamine-rich region are inactive, indicating that this region is both necessary and sufficient for recruitment of SRC1 to the androgen receptor. This recruitment is in direct contrast to the recruitment of SRC1 to the estrogen receptor, which requires interaction with the ligand-binding domain.