A model for mammalian male determination based on a passive Y chromosome

A model is suggested for mammalian male determination based on interactions postulated to occur among an autosomal repressor gene, an X-linked male-determining gene termed Tdx, and multiple copies of certain DNA sequences on the Y chromosome that do not code for any protein. The repressor, synthesised in limited amounts, has higher affinity for the Y-linked sequences than for Tdx and its affinity for Tdx is greater than that of RNA polymerase. In XY cells the Y effectively binds all available repressor, permitting transcription of Tdx to occur. In XX cells, since competition from the Y-linked high-affinity sequences is absent, the repressor binds to Tdx and prevents transcription. As a result of this competition between Tdx and the Y-linked high-affinity sites for limiting concentrations of the autosomal repressor, the product of the Tdx gene (TDX) is synthesized in the male but not in the female. It is suggested that in determination of the male sex, the role of the Y chromosome is to serve as a sink for the Tdx repressor. The proposed interactions provide a plausible explanation for the genetic properties of several anomalies of sexual development in mouse, man, and other mammals. The model suggests that the postulated multiple, highaffinity sequences on the Y chromosome of the mouse are included among the DNA sequences referred to as the Sxr-Bkm sequences.

Targeted 3' processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing.

Noncoding RNA is emerging as an important regulator of gene expression in many organisms. We are characterizing RNA-mediated chromatin silencing of the Arabidopsis major floral repressor gene, FLC. Through suppressor mutagenesis, we identify a requirement for CstF64 and CstF77, two conserved RNA 3'-end-processing factors, in FLC silencing. However, FLC sense transcript 3' processing is not affected in the mutants. Instead, CstF64 and CstF77 are required for 3' processing of FLC antisense transcripts. A specific RNA-binding protein directs their activity to a proximal antisense polyadenylation site. This targeted processing triggers localized histone demethylase activity and results in reduced FLC sense transcription. Targeted 3' processing of antisense transcripts may be a common mechanism triggering transcriptional silencing of the corresponding sense gene.

Altered interactions within FY/AtCPSF complexes required for Arabidopsis FCA-mediated chromatin silencing.

The role of RNA metabolism in chromatin silencing is now widely recognized. We have studied the Arabidopsis RNA-binding protein FCA that down-regulates an endogenous floral repressor gene through a chromatin mechanism involving histone demethylase activity. This mechanism needs FCA to interact with an RNA 3' processing/polyadenylation factor (FY/Pfs2p), but the subsequent events leading to chromatin changes are unknown. Here, we show that this FCA-FY interaction is required for general chromatin silencing roles where hairpin transgenes induce DNA methylation of an endogenous gene. We also show 2 conserved RNA processing factors, AtCPSF100 and AtCPSF160, but not FCA, are stably associated with FY in vivo and form a range of different-sized complexes. A hypomorphic fy allele producing a shorter protein, able to provide some FY functions but unable to interact with FCA, reduces abundance of some of the larger MW complexes. Suppressor mutants, which specifically disrupt the FY motif through which FCA interacts, also lacked these larger complexes. Our data support a model whereby FCA, perhaps after recognition of a specific RNA feature, transiently interacts with FY, an integral component of the canonical RNA 3' processing machinery, changing the interactions of the different RNA processing components. These altered interactions would appear to be a necessary step in this RNA-mediated chromatin silencing.

Canonical P elements are transcriptionally active in the saltans group of Drosophila

Up to now, investigations of expression and regulation of P transposable element have been almost exclusively carried out with the Drosophila melanogaster canonical P element. Analyzing eight species of the saltans group, we detected transposase mRNA in germline tissues of D. saltans and D. prosaltans and repressor mRNA in somatic tissues of D. saltans and D. sturtevanti. Sequencing analysis suggested that these transcripts might belong to the canonical subfamily and that they can be transpositionally active only in D. saltans. dN and dS values of Adh and the P element suggested that the sequences found in D. saltans and D. prosaltans might have been present in the ancestor of the saltans subgroup and that the sequence found in D. sturtevanti might have been horizontally transferred from D. saltans.

Oscillatory genetic circuits: new designs and mathematical models

Introduction and motivation: A wide variety of organisms have developed in-ternal biomolecular clocks in order to adapt to cyclic changes of the environment. Clock operation involves genetic networks. These genetic networks have to be mod¬eled in order to understand the underlying mechanism of oscillations and to design new synthetic cellular clocks. This doctoral thesis has resulted in two contributions to the fields of genetic clocks and systems and synthetic biology, generally. The first contribution is a new genetic circuit model that exhibits an oscillatory behav¬ior through catalytic RNA molecules. The second and major contribution is a new genetic circuit model demonstrating that a repressor molecule acting on the positive feedback of a self-activating gene produces reliable oscillations. First contribution: A new model of a synthetic genetic oscillator based on a typical two-gene motif with one positive and one negative feedback loop is pre¬sented. The originality is that the repressor is a catalytic RNA molecule rather than a protein or a non-catalytic RNA molecule. This catalytic RNA is a ribozyme that acts post-transcriptionally by binding to and cleaving target mRNA molecules. This genetic clock involves just two genes, a mRNA and an activator protein, apart from the ribozyme. Parameter values that produce a circadian period in both determin¬istic and stochastic simulations have been chosen as an example of clock operation. The effects of the stochastic fluctuations are quantified by a period histogram and autocorrelation function. The conclusion is that catalytic RNA molecules can act as repressor proteins and simplify the design of genetic oscillators. Second and major contribution: It is demonstrated that a self-activating gene in conjunction with a simple negative interaction can easily produce robust matically validated. This model is comprised of two clearly distinct parts. The first is a positive feedback created by a protein that binds to the promoter of its own gene and activates the transcription. The second is a negative interaction in which a repressor molecule prevents this protein from binding to its promoter. A stochastic study shows that the system is robust to noise. A deterministic study identifies that the oscillator dynamics are mainly driven by two types of biomolecules: the protein, and the complex formed by the repressor and this protein. The main conclusion of this study is that a simple and usual negative interaction, such as degradation, se¬questration or inhibition, acting on the positive transcriptional feedback of a single gene is a sufficient condition to produce reliable oscillations. One gene is enough and the positive transcriptional feedback signal does not need to activate a second repressor gene. At the genetic level, this means that an explicit negative feedback loop is not necessary. Unlike many genetic oscillators, this model needs neither cooperative binding reactions nor the formation of protein multimers. Applications and future research directions: Recently, RNA molecules have been found to play many new catalytic roles. The first oscillatory genetic model proposed in this thesis uses ribozymes as repressor molecules. This could provide new synthetic biology design principles and a better understanding of cel¬lular clocks regulated by RNA molecules. The second genetic model proposed here involves only a repression acting on a self-activating gene and produces robust oscil¬lations. Unlike current two-gene oscillators, this model surprisingly does not require a second repressor gene. This result could help to clarify the design principles of cellular clocks and constitute a new efficient tool for engineering synthetic genetic oscillators. Possible follow-on research directions are: validate models in vivo and in vitro, research the potential of second model as a genetic memory, investigate new genetic oscillators regulated by non-coding RNAs and design a biosensor of positive feedbacks in genetic networks based on the operation of the second model Resumen Introduccion y motivacion: Una amplia variedad de organismos han desarro-llado relojes biomoleculares internos con el fin de adaptarse a los cambios ciclicos del entorno. El funcionamiento de estos relojes involucra redes geneticas. El mo delado de estas redes geneticas es esencial tanto para entender los mecanismos que producen las oscilaciones como para diseiiar nuevos circuitos sinteticos en celulas. Esta tesis doctoral ha dado lugar a dos contribuciones dentro de los campos de los circuitos geneticos en particular, y biologia de sistemas y sintetica en general. La primera contribucion es un nuevo modelo de circuito genetico que muestra un comportamiento oscilatorio usando moleculas de ARN cataliticas. La segunda y principal contribucion es un nuevo modelo de circuito genetico que demuestra que una molecula represora actuando sobre el lazo de un gen auto-activado produce oscilaciones robustas. Primera contribucion: Es un nuevo modelo de oscilador genetico sintetico basado en una tipica red genetica compuesta por dos genes con dos lazos de retroa-limentacion, uno positivo y otro negativo. La novedad de este modelo es que el represor es una molecula de ARN catalftica, en lugar de una protefna o una molecula de ARN no-catalitica. Este ARN catalitico es una ribozima que actua despues de la transcription genetica uniendose y cortando moleculas de ARN mensajero (ARNm). Este reloj genetico involucra solo dos genes, un ARNm y una proteina activadora, aparte de la ribozima. Como ejemplo de funcionamiento, se han escogido valores de los parametros que producen oscilaciones con periodo circadiano (24 horas) tanto en simulaciones deterministas como estocasticas. El efecto de las fluctuaciones es-tocasticas ha sido cuantificado mediante un histograma del periodo y la función de auto-correlacion. La conclusion es que las moleculas de ARN con propiedades cataliticas pueden jugar el misnio papel que las protemas represoras, y por lo tanto, simplificar el diseno de los osciladores geneticos. Segunda y principal contribucion: Es un nuevo modelo de oscilador genetico que demuestra que un gen auto-activado junto con una simple interaction negativa puede producir oscilaciones robustas. Este modelo ha sido estudiado y validado matematicamente. El modelo esta compuesto de dos partes bien diferenciadas. La primera parte es un lazo de retroalimentacion positiva creado por una proteina que se une al promotor de su propio gen activando la transcription. La segunda parte es una interaction negativa en la que una molecula represora evita la union de la proteina con el promotor. Un estudio estocastico muestra que el sistema es robusto al ruido. Un estudio determinista muestra que la dinamica del sistema es debida principalmente a dos tipos de biomoleculas: la proteina, y el complejo formado por el represor y esta proteina. La conclusion principal de este estudio es que una simple y usual interaction negativa, tal como una degradation, un secuestro o una inhibition, actuando sobre el lazo de retroalimentacion positiva de un solo gen es una condition suficiente para producir oscilaciones robustas. Un gen es suficiente y el lazo de retroalimentacion positiva no necesita activar a un segundo gen represor, tal y como ocurre en los relojes actuales con dos genes. Esto significa que a nivel genetico un lazo de retroalimentacion negativa no es necesario de forma explicita. Ademas, este modelo no necesita reacciones cooperativas ni la formation de multimeros proteicos, al contrario que en muchos osciladores geneticos. Aplicaciones y futuras lineas de investigacion: En los liltimos anos, se han descubierto muchas moleculas de ARN con capacidad catalitica. El primer modelo de oscilador genetico propuesto en esta tesis usa ribozimas como moleculas repre¬soras. Esto podria proporcionar nuevos principios de diseno en biologia sintetica y una mejor comprension de los relojes celulares regulados por moleculas de ARN. El segundo modelo de oscilador genetico propuesto aqui involucra solo una represion actuando sobre un gen auto-activado y produce oscilaciones robustas. Sorprendente-mente, un segundo gen represor no es necesario al contrario que en los bien conocidos osciladores con dos genes. Este resultado podria ayudar a clarificar los principios de diseno de los relojes celulares naturales y constituir una nueva y eficiente he-rramienta para crear osciladores geneticos sinteticos. Algunas de las futuras lineas de investigation abiertas tras esta tesis son: (1) la validation in vivo e in vitro de ambos modelos, (2) el estudio del potential del segundo modelo como circuito base para la construction de una memoria genetica, (3) el estudio de nuevos osciladores geneticos regulados por ARN no codificante y, por ultimo, (4) el rediseno del se¬gundo modelo de oscilador genetico para su uso como biosensor capaz de detectar genes auto-activados en redes geneticas.

A soil-based microbial biofilm exposed to 2,4-D: bacterial community development and establishment of conjugative plasmid pJP4

A soil suspension was used as a source to initiate the development of microbial communities in flow cells irrigated with 2,4-dichlorophenoxyacetic acid (2,4-D) (25 mu g ml(-1)). Culturable bacterial members of the community were identified by 16S rRNA gene sequencing and found to be members of the genera Pseudomonas, Burkholderia, Collimonas and Rhodococcus. A 2,4-D degrading donor strain, Pseudomonas putida SM 1443 (pJP4::gfp), was inoculated into flow cell chambers containing 2-day old biofilm communities. Transfer of pJP4::gfp from the donor to the bacterial community was detectable as GFP fluorescing cells and images were captured using confocal scanning laser microscopy (GFP fluorescence was repressed in the donor due to the presence of a chromosomally located lacl(q) repressor gene). Approximately 5-10 transconjugant microcolonies, 20-40 mu m in diameter, could be seen to develop in each chamber. A 2,4-D degrading transconjugant strain was isolated from the flow cell system belonging to the genus Burkholderia.

Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EMSAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903–10907]. All cell-free extracts formed high-molecular weight protein–DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein–DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA–protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a “mycoparasitic” protein–protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein–DNA complex.

Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells

Introduction Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. Methods MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann–Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). Results Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. Conclusions This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time

SVP-like MADS domain transcription factors have been shown to regulate flowering time and both inflorescence and flower development in annual plants, while having effects on growth cessation and terminal bud formation in perennial species. Previously, four SVP genes were described in woody perennial vine kiwifruit (Actinidia spp.), with possible distinct roles in bud dormancy and flowering. Kiwifruit SVP3 transcript was confined to vegetative tissues and acted as a repressor of flowering as it was able to rescue the Arabidopsis svp41 mutant. To characterize kiwifruit SVP3 further, ectopic expression in kiwifruit species was performed. Ectopic expression of SVP3 in A. deliciosa did not affect general plant growth or the duration of endodormancy. Ectopic expression of SVP3 in A. eriantha also resulted in plants with normal vegetative growth, bud break, and flowering time. However, significantly prolonged and abnormal flower, fruit, and seed development were observed, arising from SVP3 interactions with kiwifruit floral homeotic MADS-domain proteins. Petal pigmentation was reduced as a result of SVP3-mediated interference with transcription of the kiwifruit flower tissue-specific R2R3 MYB regulator, MYB110a, and the gene encoding the key anthocyanin biosynthetic step, F3GT1. Constitutive expression of SVP3 had a similar impact on reproductive development in transgenic tobacco. The flowering time was not affected in day-neutral and photoperiod-responsive Nicotiana tabacum cultivars, but anthesis and seed germination were significantly delayed. The accumulation of anthocyanin in petals was reduced and the same underlying mechanism of R2R3 MYB NtAN2 transcript reduction was demonstrated.

Failure to launch: The self-regulating Md -MYB10R6 gene from apple is active in flowers but not leaves of Petunia

Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples (Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md-MYB10R6, are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md-MYB10R6 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md-MYB10R6 transgene (MYB10–R6pro:MYB10:MYB10term) activated anthocyanin synthesis when transiently expressed in Antirrhinumroseadorsea petals and petunia leaf discs. Stable transgenic petunias containing Md-MYB10R6 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of ‘Mitchell’ petunia. The absence of foliar pigmentation was due to the failure of the Md-MYB10R6 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md-MYB10R6 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals.

Mammalian Mat1 subunit of Transcription Factor IIH in gene-specific and general transcription

All protein-encoding genes in eukaryotes are transcribed into messenger RNA (mRNA) by RNA Polymerase II (RNAP II), whose activity therefore needs to be tightly controlled. An important and only partially understood level of regulation is the multiple phosphorylations of RNAP II large subunit C-terminal domain (CTD). Sequential phosphorylations regulate transcription initiation and elongation, and recruit factors involved in co-transcriptional processing of mRNA. Based largely on studies in yeast models and in vitro, the kinase activity responsible for the phosphorylation of the serine-5 (Ser5) residues of RNAP II CTD has been attributed to the Mat1/Cdk7/CycH trimer as part of Transcription Factor IIH. However, due to the lack of good mammalian genetic models, the roles of both RNAP II Ser5 phosphorylation as well as TFIIH kinase in transcription have provided ambiguous results and the in vivo kinase of Ser5 has remained elusive. The primary objective of this study was to elucidate the role of mammalian TFIIH, and specifically the Mat1 subunit in CTD phosphorylation and general RNAP II-mediated transcription. The approach utilized the Cre-LoxP system to conditionally delete murine Mat1 in cardiomyocytes and hepatocytes in vivo and and in cell culture models. The results identify the TFIIH kinase as the major mammalian Ser5 kinase and demonstrate its requirement for general transcription, noted by the use of nascent mRNA labeling. Also a role for Mat1 in regulating general mRNA turnover was identified, providing a possible rationale for earlier negative findings. A secondary objective was to identify potential gene- and tissue-specific roles of Mat1 and the TFIIH kinase through the use of tissue-specific Mat1 deletion. Mat1 was found to be required for the transcriptional function of PGC-1 in cardiomyocytes. Transriptional activation of lipogenic SREBP1 target genes following Mat1 deletion in hepatocytes revealed a repressive role for Mat1apparently mediated via co-repressor DMAP1 and the DNA methyltransferase Dnmt1. Finally, Mat1 and Cdk7 were also identified as a negative regulators of adipocyte differentiation through the inhibitory phosphorylation of Peroxisome proliferator-activated receptor (PPAR) γ. Together, these results demonstrate gene- and tissue-specific roles for the Mat1 subunit of TFIIH and open up new therapeutic possibilities in the treatment of diseases such as type II diabetes, hepatosteatosis and obesity.

Silencing of toxic gene expression by Fis

Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.