Transcriptional and post-transcriptional regulation of SPAST, the gene most frequently mutated in hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70-80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3'-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP.

TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression.

Frequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage-independent growth in vitro, and loss of TMEM16A resulted in inhibition of tumor growth both in vitro and in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal-regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide a mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN.

Evolution and variation of the nifD and hupL elements in the heterocystous cyanobacteria.

In heterocystous cyanobacteria, heterocyst differentiation is accompanied by developmentally regulated DNA rearrangements that occur within the nifD and hupL genes, referred to as the nifD and hupL elements. These elements are segments of DNA that are embedded within the coding region of each gene and range from 4 to 24 kb in length. The nifD and hupL elements are independently excised from the genome during the later stages of differentiation by the site-specific recombinases, XisA and XisC, respectively, which are encoded within the elements themselves. Here we examine the variation and evolution of the nifD and hupL elements by comparing full-length nifD and hupL element sequences and by phylogenetic analysis of xisA and xisC gene sequences. There is considerable variation in the size and composition of the nifD and hupL elements, however, conserved regions are also present within representatives of each element. The data suggest that the nifD and hupL elements have undergone a complex pattern of insertions, deletions, translocations and sequence divergence over the course of evolution, but that conserved regions remain.

Decreased expression of miR-125b and miR-100 in oral cancer cells contributes to malignancy.

Altered microRNA (miRNA) expression profiles have been observed in numerous malignancies, including oral squamous cell carcinoma (OSCC). However, their role in disease is not entirely clear. Several genetic aberrations are characteristic of OSCC, with amplification of chromosomal band 11q13 and loss of distal 11q being among the most prevalent. It is not known if the expression levels of miRNAs in these regions are altered or whether they play a role in disease. We hypothesize that the expression of miRNAs mapping to 11q are altered in OSCC because of loss or amplification of chromosomal material, and that this contributes to the development and progression of OSCC. We found that miR-125b and miR-100 are down-regulated in OSCC tumor and cell lines, and that transfecting cells with exogenous miR-125b and miR-100 significantly reduced cell proliferation and modified the expression of target and nontarget genes, including some that are overexpressed in radioresistant OSCC cells. In conclusion, the down-regulation of miR-125b and miR-100 in OSCC appears to play an important role in the development and/or progression of disease and may contribute to the loss of sensitivity to ionizing radiation.

Characterization of a 4 kb variant of the nifD element in Anabaena sp. strain ATCC 33047.

Heterocyst differentiation in some cyanobacteria is accompanied by a programmed DNA rearrangement within the nitrogen fixation gene nifD. The nifD element is excised from within nifD during the latter stages of heterocyst differentiation by site-specific recombination. There is considerable variation in those nifD elements examined thus far, with Nostoc sp. Strain PCC 7120 and Anabaena variabilis having 11 kb elements, and Nostoc punctiforme having a 24 kb element. Here we characterize a 4 kb nifD element in Anabaena sp. Strain ATCC 33047, and compare it with the other sequenced nifD elements. While there is considerable variation in both the size (ranging from 4 kb to 24 kb) and composition of the nifD elements examined thus far, there are regions that are conserved in all. These conserved regions include the flanking 3' and 5' regions, the xisA gene, and a small open reading frame known as ORF2 in Nostoc sp. Strain PCC 7120.

The evolutionary history of nitrogen fixation, as assessed by NifD.

The evolutionary history of nitrogen fixation has been vigorously debated for almost two decades. Previous phylogenetic analyses of nitrogen fixation genes (nif) have shown support for either evolution by vertical descent or lateral transfer, depending on the specific nif gene examined and the method of analyses used. The debate centers on the placement and monophyly of the cyanobacteria, proteobacteria, and Gram-positive bacteria (actinobacteria and firmicutes). Some analyses place the cyanobacteria and actinobacteria within the proteobacteria, which suggests that the nif genes have been laterally transferred since this topology is incongruent with ribosomal phylogenies, the standard marker for comparison. Other nif analyses resolve and support the monophyly of the cyanobacteria, proteobacteria, and actinobacteria, supporting vertical descent. We have revisited these conflicting scenarios by analyzing nifD from an increased number of cyanobacteria, proteobacteria, and Gram-positive bacteria. Parsimony analyses of amino acid sequences and maximum likelihood analysis of nucleic acid sequences support the monophyly of the cyanobacteria and actinobacteria but not the proteobacteria, lending support for vertical descent. However, distance analysis of nucleic acid sequences placed the actinobacteria within the proteobacteria, supporting lateral transfer. We discuss evidence for both vertical descent and lateral transfer of nitrogen fixation.

Molecular phylogeny of the heterocystous cyanobacteria (subsections IV and V) based on nifD.

The heterocystous cyanobacteria are currently placed in subsections IV and V, which are distinguished by cellular division in one plane (false branching) and in more than one plane (true branching), respectively. Published phylogenies of 16S rRNA gene sequence data support the monophyly of the heterocystous cyanobacteria, with members of subsection V embedded within subsection IV. It has been postulated that members of subsection V arose from within subsection IV. Therefore, phylogenetic analysis of nucleotide sequences of the nitrogen-fixation gene nifD from representatives of subsections IV and V was performed by using maximum-likelihood criteria. The heterocystous cyanobacteria are supported as being monophyletic, with the non-heterocystous cyanobacteria as their closest relative. However, neither subsection IV nor subsection V is monophyletic, with representatives of both subsections intermixed in two sister clades. Analysis of nifD does not support recognition of two distinct subsections.

Molecular differentiation of the heterocystous cyanobacteria, Nostoc and Anabaena, based on complete NifD sequences.

The segregation of Nostoc and Anabaena into separate genera has been debated for some time. The nitrogen fixation gene nifD was completely sequenced from representatives of these genera and analyzed phylogenetically, by using the representatives of other genera of the heterocystous cyanobacteria as outgroups. We were clearly able to differentiate between Nostoc and Anabaena in all analyses used. Our data suggest that Nostoc and Anabaena should remain as separate genera.