Proximal cis-acting elements cooperate to set Hoxb-7 (Hox-2.3) expression boundaries in transgenic mice

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 71-82 ◽  
Author(s):  
R. Vogels ◽  
J. Charite ◽  
W. de Graaff ◽  
J. Deschamps
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Hox Genes ◽  
Gene Clustering ◽  
Lacz Gene ◽  
Dependent Manner ◽  
Endogenous Gene ◽  
Cis Acting ◽  
Gene Map ◽  
Partial Overlap

The Hox genes have been proved to be instrumental in establishing the positional identity of cells along the embryonic anteroposterior (A-P) axis. Studying the regulation of these genes is a first step toward elucidating the molecular basis of regionalization during embryogenesis. We report here on the identification of cis-acting elements controlling the expression of Hoxb-7 (Hox-2.3). We show that elements driving A-P restricted gene expression are located within the 3.5 kb proximal upstream sequences of the Hoxb-7 gene. A deletion analysis provides evidence for at least three cis-acting control elements upstream from Hoxb-7, and for cooperative interactions between some of these elements in generating the A-P restricted transgenic pattern. One element, conferring by itself Hox-like expression boundaries to the transgene, has been studied in more detail and found to act in an orientation-and promoter-dependent manner. Together the 3.5 kb sequences proximal to Hoxb-7 mediate A-P restricted Hoxb-7/lacZ gene expression in a domain showing rostral boundaries more posterior than those of Hoxb-7. The evolution throughout embryogenesis of the expression pattern of a transgene carrying these sequences has been analysed and shown to mimick that of the endogenous gene, except for a slight delay in the initial expression. We conclude that the transgenes that we tested, spanning a total of 27 kb genomic sequences, do not reproduce all the features of the Hoxb-7 expression pattern. The differences in expression between Hoxb-7 and the transgenes may reveal an aspect of the Hox regulation for which either remote cis-acting control elements and/or gene clustering is required. Additional features that may have favoured maintenance of clustered organisation during evolution are partial overlap of transcription units with the regulatory regions of the neighbouring genes, and cis-regulatory interactions between multiple Hox genes: not only do cis-acting control elements of the Hoxb-7 gene map in the 3′ untranslated sequences of the Hoxb-8 (Hox-2.4) gene, but our experiments suggest that Hoxb-7 control sequences modulate expression of the Hoxb-8 gene as well.

Homeobox genes and models for patterning the hindbrain and branchial arches

Development ◽  
1991 ◽  
Vol 113 (Supplement_1) ◽  
pp. 187-196 ◽  
Author(s):  
Paul Hunt ◽  
Jenny Whiting ◽  
Ian Muchamore ◽  
Heather Marshall ◽  
Robb Krumlauf
Keyword(s):  
Gene Expression ◽  
Neural Crest ◽  
Expression Pattern ◽  
Hox Genes ◽  
Homeobox Genes ◽  
Neural Plate ◽  
The Body ◽  
Hox Gene ◽  
Hox Code ◽  
Branchial Region

Antennapedia class homeobox genes, which in insects are involved in regional specification of the segmented central regions of the body, have been implicated in a similar role in the vertebrate hindbrain. The development of the hindbrain involves the establishment of compartments which are subsequently made distinct from each other by Hox gene expression, implying that the lineage of neural cells may be an important factor in their development. The hindbrain produces the neural crest that gives rise to the cartilages of the branchial skeleton. Lineage also seems to be important in the neural crest, as experiments have shown that the crest will form cartilages appropriate to its level of origin when grafted to a heterotopic location. We show how the Hox genes could also be involved in patterning the mesenchymal structures of the branchial skeleton. Recently it has been proposed that the rhombomererestricted expression pattern of Hox 2 genes is the result of a tight spatially localised induction from underlying head mesoderm, in which a prepattern of Hox expression is visible. We find no evidence for this model, our data being consistent with the idea that the spatially localised expression pattern is a result of segmentation processes whose final stages are intrinsic to the neural plate. We suggest the following model for patterning in the branchial region. At first a segment-restricted code of Hox gene expression becomes established in the neuroepithelium and adjacent presumptive neural crest. This expression is then maintained in the neural crest during migration, resulting in a Hox code in the cranial ganglia and branchial mesenchyme that reflects the crest's rhombomere of origin. The final stage is the establishment of Hox 2 expression in the surface ectoderm which is brought into contact with neural crest-derived branchial mesenchyme. The Hox code of the branchial ectoderm is established later in development than that of the neural plate and crest, and involves the same combination of genes as the underlying crest. Experimental observations suggest the idea of an instructive interaction between branchial crest and its overlying ectoderm, which would be consistent with our observations. The distribution of clusters of Antennapedia class genes within the animal kingdom suggests that the primitive chordates ancestral to vertebrates had at least one Hox cluster. The origin of the vertebrates is thought to have been intimately linked to the appearance of the neural crest, initially in the branchial region. Our data are consistent with the idea that the branchial region of the head arose in evolution before the more anterior parts, the development of the branchial region employing the Hox genes in a more determinate patterning system. In this scenario, the anterior parts of the head arose subsequently, which may explain the greater importance of interactions in their development, and the fact that Antennapedia class Hox genes are not expressed there.

HOX Genes Expression Pattern Is Related to Phenotypical and Genotypical Subgroups but Not to Treatment Response in Pediatric ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1288-1288
Author(s):  
Julia Starkova ◽  
Blanka Vicenova ◽  
Roman Krejci ◽  
Harry A. Drabkin ◽  
Jan Trka
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Expression Patterns ◽  
Hox Gene ◽  
Age Related ◽  
Genes Expression ◽  
Significant Difference

Abstract Abstract 1288 Poster Board I-310 Homeodomain (HOX) genes encode transcription factors important for embryonic development. They are involved in normal hemopoiesis regulation and likely also in leukemogenesis as a result of translocations and other aberrations present in leukemias. In previous work Drabkin et al. demonstrated that HOX gene expression patterns differentiate major cytogenetic groups in acute myeloid leukemias. In this study we focused on HOX gene expression in pediatric acute lymphoblastic leukemias (ALL). We were interested if certain HOX genes or expression pattern could distinguish subpopulations of ALL. We analyzed the expression pattern of 21 HOX genes from HOXA and HOXB clusters and non-cluster HOX genes, CDX1 and CDX2 using qRT-PCR approach. We looked at 54 patients chosen according to phenotypic (T-ALL, BCP-ALL), prognostic (PGR – prednisone good responders, PPR – prednisone poor responders) and genotypic (BCR/ABL, MLL/AF4, TEL/AML1, hyperdiploid) characteristics. Overall analysis comparing all studied groups showed that HOXA7 (Kruskal-Wallis test p=0.000045), HOXA3 (p=0.000098), HOXB3 (p=0.00015), HOXA4 (p=0.000619) and HOXB4 (p=0.001925) genes were differently expressed among groups. Wilcoxon signed-rank test, a non-parametric statistical analysis comparing two groups against each other, showed that HOXA3, A4 and B3 distinguish BCP-ALL (w/o fusion gene) and T-ALL. Interestingly, particular HOX genes expression showed significant difference among the groups: HOXA7 gene is significantly downregulated in hyperdiploid ALL (p=0.03) compared to all other subgroups. Furthermore, HOXB7 gene is specifically upregulated in TEL/AML-positive patients (p=0.0048 vs BCP-ALL w/o fusion gene) and CDX2 is downregulated in BCR/ABL-positive patients (p=0.001 vs hyperdiploid; p=0.006 vs TEL/AML1; p=0.03 vs MLL/AF4). Suprisingly, TEL/AML1-positive patients have similar expression of HOXA1-A4 as T-ALL patients. HOX genes expression pattern seemed to differ in MLL/AF4-positive patients according to the age at diagnosis. Three patients younger than 2 months at presentation clustered together in clear contrast to the MLL/AF4-positive patient diagnosed at the age of 13 years with secALL who presented with very low overall expression of all HOX genes. Next, we looked for diversity and similarity between groups. We determined how many HOX genes were expressed differently (p<0.05) and similarly (p=1.0) between particular ALL subtypes. The most outlying couples were T-ALL vs PPR (11 genes differently expressed), T-ALL vs PGR (9 genes) and T-ALL vs TEL/AML1 (6 genes). In contrast, the closest groups were BCR/ABL vs PPR, MLL/AF4 vs T-ALL and MLL/AF4 vs PPR. Our data demonstrate that BCP-ALL (w/o known fusion gene) can be distinguished from T-ALL by the HOX gene expression (in particular HOXA3, HOXB3, HOXA4). Like in AML, expression pattern differs also among the major cytogenetical subgroups of ALL. On the other hand, within the BCP-ALL subgroup, no expression difference was found between patients with good (PGR) and poor (PPR) response to the initial steroid therapy which is known to be an excellent predictor of outcome. HOX genes of interest emerged from our analysis: low expression of HOXA7 in hyperdiploid ALL, highly expressed HOXB7 in TEL/AML1-positive ALL and specifically downregulated CDX2 in BCR/ABL-positive ALL. Age-related differences in expression in MLL/AF4-positive ALL seem to link the expression pattern rather with the relative maturity of the cell undergoing (pre)malignant transformation than with the specific changes caused by the leukemogenesis itself. This hypothesis must be tested in comparison to the HOX genes expression in sorted subtypes of normal T and B precursors. This work was supported by MSM0021620813, IGA NR/9526 and GACR 301/08/P532. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of the Saccharomyces cerevisiae general regulatory factor CP1 in methionine biosynthetic gene transcription.

1995 ◽  
Vol 15 (4) ◽  
pp. 1879-1888 ◽  
Author(s):  
K F O'Connell ◽  
Y Surdin-Kerjan ◽  
R E Baker
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Gene Transcription ◽  
Chromatin Structure ◽  
Micrococcal Nuclease ◽  
Regulatory Factor ◽  
Lacz Reporter ◽  
Endogenous Gene ◽  
Cis Acting ◽  
Nuclease Digestion

Saccharomyces cerevisiae general regulatory factor CP1 (encoded by the gene CEP1) is required for optimal chromosome segregation and methionine prototrophy. MET16-CYC1-lacZ reporter constructs were used to show that MET16 5'-flanking DNA contains a CP1-dependent upstream activation sequence (UAS). Activity of the UAS required an intact CP1-binding site, and the effects of cis-acting mutations on CP1 binding and UAS activity correlated. In most respects, MET16-CYC1-lacZ reporter gene expression mirrored that of chromosomal MET16; however, the endogenous gene was found to be activated in response to amino acid starvation (general control). The latter mechanism was both GCN4 and CP1 dependent. MET25 was also found to be activated by GCN4, albeit weakly. More importantly, MET25 transcription was strongly CP1 dependent in gcn4 backgrounds. The modulation of MET gene expression by GCN4 can explain discrepancies in the literature regarding CP1 dependence of MET gene transcription. Lastly, micrococcal nuclease digestion and indirect end labeling were used to analyze the chromatin structure of the MET16 locus in wild-type and cep1 cells. The results indicated that CP1 plays no major role in configuring chromatin structure in this region, although localized CP1-specific differences in nuclease sensitivity were detected.

Leukemic Pattern Of HOX Gene Expression Is Driven By Genetic Aberrations Through Epigenetic Modifiers

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2504-2504
Author(s):  
Julia Starkova ◽  
Karolina Kramarzova ◽  
Karel Fiser ◽  
Ester Mejstrikova ◽  
Katerina Rejlova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Hox Genes ◽  
De Novo ◽  
In Vitro Experiments ◽  
Genetic Aberrations ◽  
Hox Gene ◽  
Epigenetic Modifiers ◽  
Fab Classification

Abstract Introduction Homeobox (HOX) genes encode transcription factors crucial in embryogenesis. They are often dysregulated in malignancies including leukemias. The aberrant HOX gene expression and its regulation in leukemic cells is neither completely described nor understood. Aims Our main aim was to determine whether the leukemic HOX gene expression pattern is driven by differentiation stage of hematopoietic cells or determined de novo during the process of malignant transformation. Consequentially, we aimed to study the role epigenetic modifiers in regulation of HOX gene expression in normal and malignant hematopoiesis. Methods The expression pattern of HOX genes (cluster of HOX A and B) and epigenetic modifiers (DNMT1, DNMT3a, DNMT3b, EZH2, BMI-1, MLL, JMJD3, UTX) was assessed by qPCR in 8 FACS-sorted subpopulations of healthy BM representing stages of myeloid differentiation (each sample representing a pool of cells sorted from five individuals). The leukemic expression pattern of these genes was analyzed in diagnostic BM samples of childhood AML patients with typical genotypic and morphological (FAB classification) characteristics (N=46). In vitro experiments were performed using NB4 cell line. Results As expected HOX genes were gradually downregulated during normal differentiation of granulocytic and monocytic lineages (assessed in four consecutive differentiation stages for each lineage). In AML samples, HOX gene expression patterns differed significantly among morphological subtypes. However, HOX gene expression did not correlate among subtypes of AML and their physiologically differentiated counterparts. Interestingly, unsupervised hierarchical clustering (HCA) divided AML patients into four main clusters characterized by the presence of prevalent gene rearrangement (PML-RARa, AML1-ETO, MLL rearrangements and NK-AML). The presence of PML/RARa rearrangement was strongly associated with the lowest expression of both HOXA and HOXB clusters, while the other groups had more variable expression of HOX genes. Moreover, the effect of genetic aberrations on HOX gene expression was clearly apparent within AML M2 and M4 subtypes, where AML1/ETO+ or CBFb/MYH11+ patients had significantly lower expression of HOX genes compared to patients with the same FAB classification but without the rearrangements. The expression pattern of epigenetic modifiers in sorted subpopulations of healthy BM followed their expected role in transcriptional regulation during differentiation. However, there was no relation of this pattern to HOX gene expression. On the contrary, in AML samples, the expression levels of epigenetic modifiers clearly correlated with expression profile of HOX genes. These results were supported by unsupervised HCA based on the expression of epigenetic modifiers that showed upregulation of histon demethylases JMJD3 and UTX together with downregulation of DNMT3b in concordance with high levels of HOX genes. Negative correlation between JMJD3 and DNMT3b expression was observed in all leukemic samples (p=0.03); most apparently in PML/RARa+ patients. Therefore we further studied the impact of genetic aberrations on the epigenetic regulation of HOX gene expression in vitrowith PML-RARa+ cell line. Treatment of NB4 cells with ATRA (8, 24hours, 1uM, 10uM) increased the levels of particular HOX genes (HOXA5, A7, B4, B7; FCA=2.8; 1.7; 4; 4 respectively) as well as JMJD3 (FCA=3) and UTX (FCA=1.6). Concordantly, the expression of DNMT3b (FCA=5) was downregulated. The hypothetical driving effect of PML-RARa on de novo determination of leukemic HOX gene expression is further supported by our Results. PML-RARa+ patients had the lowest HOX gene expression regardless of their FLT3/ITD status – previously shown to upregulate strongly HOX genes expression. Conclusion We conclude that the leukemic expression pattern of HOX genes does not reflect the differentiation stages of malignant cells. Our data also demonstrate different contribution of epigenetic modifiers to the HOX gene expression in healthy and malignant hematopoiesis. Moreover, HCA and expression data together with the results of in vitro experiments suggest that the specific molecular aberrations (as exemplified by PML-RARa) participate in regulation of leukemic HOX gene expression through epigenetic changes. Supported by GACR P304/12/2214, GAUK 568213, 00064203. Disclosures: No relevant conflicts of interest to declare.

scholarly journals X-ray 3D imaging of gene expression in whole-mount murine brain by microCT, implication for functional analysis of tRNA endonuclease 54 gene mutated in pontocerebellar hypoplasia

2019 ◽  
Author(s):  
Olga Ermakova ◽  
Tiziana Orsini ◽  
Paolo Fruscoloni ◽  
Francesco Chiani ◽  
Alessia Gambadoro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Reporter Gene ◽  
Three Dimensional ◽  
Gene Expression Pattern ◽  
Lacz Gene ◽  
Pontocerebellar Hypoplasia ◽  
X Ray ◽  
Whole Mount ◽  
Murine Brain

AbstractAcquisition of detailed structural and molecular information from intact biological samples, while preserving cellular three-dimensional structures, still represents a challenge for biological studies aiming to unravel system functions. Here we describe a novel X-ray-based methodology for analysis of gene expression pattern in intact murine brain ex vivo by microCT. The method relays on detection of bromine molecules in the products of enzymatic reaction generated by the β-galactosidase (lacZ) gene reporter. To demonstrate the feasibility of the method, the analysis of the expression pattern of tRNA endonuclease 54 (Tsen54)-lacZ reporter gene in the whole-mount murine brain in semi-quantitative manner is performed. Mutations in Tsen54 gene causes pontocerebellar hypoplasia (PCH), severe neurodegenerative disorder with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we have demonstrated that highest Tsen54 expression observed in anatomical brain substructures important for the normal motor and memory functions in mice. In the forebrain strong expression in perirhinal, retrosplenial and secondary motor areas was observed. In olfactory area Tsen54 is highly expressed in the nucleus of the lateral olfactory tract, anterior olfactory and bed nuclei, while in hypothalamus in lateral mammillary nucleus and preoptic area. In hindbrain Tsen54 is expressed in the reticular, cuneate and trigeminal nuclei of medulla, and in pontine gray of pons and in cerebellum, in the molecular and Purkinje cell layers. Delineating anatomical brain regions in which Tsen54 is strongly expressed will allow functionally address the role Tsen54 gene in normal physiology and in PCH disease.Significance StatementCharacterization of gene expression pattern in the brain of model organisms is critical for unravelling the gene function in normal physiology and disease. It is performed by optical imaging of the two-dimensional brain sections which then assembled in volume images. Here we applied microCT platform, which allows three-dimensional imaging of non transparent samples, for analysis of gene expression. This method based on detection by X-ray the bromine molecules presented in the products generated by enzymatic activity of b-galactosidase reporter gene. With this method we identify anatomical brain substructures in which Tsen54 gene, mutated in pontocerebellar hypoplasia disease, is expressed.

scholarly journals Flow-dependent regulation of endothelial Tie2 by GATA3 in vivo

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Temitayo O. Idowu ◽  
Valerie Etzrodt ◽  
Thorben Pape ◽  
Joerg Heineke ◽  
Klaus Stahl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Experimental Models ◽  
Common Denominator ◽  
Dependent Manner ◽  
Pulmonary Endothelium ◽  
Delivery Platform ◽  
Transient Overexpression ◽  
Plasmid Delivery

Abstract Background Reduced endothelial Tie2 expression occurs in diverse experimental models of critical illness, and experimental Tie2 suppression is sufficient to increase spontaneous vascular permeability. Looking for a common denominator among different critical illnesses that could drive the same Tie2 suppressive (thereby leak inducing) phenotype, we identified “circulatory shock” as a shared feature and postulated a flow-dependency of Tie2 gene expression in a GATA3 dependent manner. Here, we analyzed if this mechanism of flow-regulation of gene expression exists in vivo in the absence of inflammation. Results To experimentally mimic a shock-like situation, we developed a murine model of clonidine-induced hypotension by targeting a reduced mean arterial pressure (MAP) of approximately 50% over 4 h. We found that hypotension-induced reduction of flow in the absence of confounding disease factors (i.e., inflammation, injury, among others) is sufficient to suppress GATA3 and Tie2 transcription. Conditional endothelial-specific GATA3 knockdown (B6-Gata3tm1-Jfz VE-Cadherin(PAC)-cerERT2) led to baseline Tie2 suppression inducing spontaneous vascular leak. On the contrary, the transient overexpression of GATA3 in the pulmonary endothelium (jet-PEI plasmid delivery platform) was sufficient to increase Tie2 at baseline and completely block its hypotension-induced acute drop. On the functional level, the Tie2 protection by GATA3 overexpression abrogated the development of pulmonary capillary leakage. Conclusions The data suggest that the GATA3–Tie2 signaling pathway might play a pivotal role in controlling vascular barrier function and that it is affected in diverse critical illnesses with shock as a consequence of a flow-regulated gene response. Targeting this novel mechanism might offer therapeutic opportunities to treat vascular leakage of diverse etiologies.

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