Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.     

Functional comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 homeoboxes

A number of models attempt to explain the functional relationships of Hox genes. The functional equivalence model states that mammalian Hox-encoded proteins are largely functionally equivalent, and that Hox quantity is more important than Hox quality. In this report, we describe the results of two homeobox swaps. In one case, the homeobox of Hoxa 11 was replaced with that of the very closely related Hoxa 10. Developmental function was assayed by analyzing the phenotypes of all possible allele combinations, including the swapped allele, and null alleles for Hoxa 11 and Hoxd 11. This chimeric gene provided wild-type function in the development of the axial skeleton and male reproductive tract, but served as a hypomorph allele in the development of the appendicular skeleton, kidneys, and female reproductive tract. In the other case, the Hoxa 11 homeobox was replaced with that of the divergent Hoxa 4 gene. This chimeric gene provided near recessive null function in all tissues except the axial skeleton, which developed normally. These results demonstrate that even the most conserved regions of Hox genes, the homeoboxes, are not functionally interchangeable in the development of most tissues. In some cases, developmental function tracked with the homeobox, as previously seen in simpler organisms. Homeoboxes with more 5' cluster positions were generally dominant over more 3' homeoboxes, consistent with phenotypic suppression seen in Drosophila. Surprisingly, however, all Hox homeoboxes tested did appear functionally equivalent in the formation of the axial skeleton. The determination of segment identity is one of the most evolutionarily ancient functions of Hox genes. It is interesting that Hox homeoboxes are interchangeable in this process, but are functionally distinct in other aspects of development.     

An oviduct-specific and enhancer-like element resides at about -3000 in the chicken ovalbumin gene

The chicken ovalbumin (Ov) gene is one of the best models to study tissue-specific gene regulation because it is only expressed in the oviduct. In this paper, a tissue-specific element was characterized by 5'-flanking region deletion in combination with in vivo gene electroporation (EP). Plasmids with varying lengths of truncated Ov 5'-flanking region fused to the Renilla luciferase reporter gene were transfected in vivo into oviduct, muscle, and pancreas. A chicken oviduct-specific and enhancer-like region (designated COSE) was implicated between -3100 and -2800. The COSE showed up-regulation of gene expression in oviduct, but not in muscle or in pancreas. The COSE region was further characterized by gel mobility shift assays using nuclear extracts from oviduct, pancreas and liver. With the region from -2900 to -2851, designated T2, there were two distinct protein-DNA complexes: one found only in oviduct extract and the other detected only in pancreas and liver. These data suggest a model where the regulation of Ov gene expression in the oviduct and non-oviduct tissues is exerted at least in part by the presence of protein modulators that bind to the COSE element.     

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.     

Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, we generated mice simultaneously mutant at both loci. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified rhombomeres.     

The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues

The octopine synthase (ocs or ocs-like) element has been previously reported to be responsive to the plant hormones, auxin, salicylic acid, and methyl jasmonate. Using transient assays with carrot protoplasts, we have demonstrated that an ocs element from the soybean auxin-inducible GH2/4 promoter is not only activated by strong auxins (i.e, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, -naphthalene acetic acid) and salicylic acid, but also by weak auxin analogues (-naphthalene acetic acid), inactive auxin analogs (i.e., 2,3-dichlorophenoxyacetic acid, 2,4,6-trichlorophenoxyacetic acid), and inactive salicylic acid analogs (3-hydroxybenzoic acid and 4-hydroxybenzoic acid). Our results indicate that the ocs element in the GH2/4 promoter is not selectively induced by plant hormones and might function similarly to tandem AP-1 sites in some animal glutathione S-transferase (GST) genes. The ocs element, like the AP-1 sites in animal GST promoters, may be induced not only by certain hormones but also by some non-hormonal stress-inducing or electrophilic agents.Abbreviations GST glutathione S-transferase - MUG 4-methyl-umbelliferyl-glucuronide - GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - NAA naphthalene acetic acid - SA salicylic acid - SARE putative salicylic acid-responsive element - BA benzoic acid - UTR untranslated region - nos nopaline synthase - ocs octopine synthase - mas mannopine synthase - ocs element-(–)46 CaMV 35S promoter-GUS reporter gene: the ocs element fused to a minimal –46 cauliflower mosaic virus 35S promoter fused to a GUS reporter gene with a 3 nos untranslated region     

An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2

The authors note that P values presented in the original Fig 1A and Appendix Fig S1A were not assessed using a proper statistical analysis method. In contrast to the initially employed two‐group t‐test, a one‐sample one‐tailed t‐test is appropriate here, as the basic null hypothesis is that the proportion of MT FOXL2 mRNA in each AGCT patient is lower than WT {H ₀: WT(%) > MT(%) }. New p values are presented in the corrected Fig 1A and Appendix Fig S1A, which are P < 0.00001 and P < 0.05, respectively. These revised P values did not affect the conclusion drawn.     

An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult‐type granulosa cell tumors (AGCTs), introduces a target site for miR‐1236, which causes haploinsufficiency of the tumor‐suppressor FOXL2. This miR‐1236‐mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA‐loaded RNA‐induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR‐1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation‐mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA‐targeting disease‐associated mutations in the CDS by forming a non‐canonical miRISC.     

A point mutation in the VP4 coding sequence of coxsackievirus B4 influences virulence.

In analyzing the molecular determinants of virulence of coxsackievirus B4, chimeric viruses were constructed from avirulent and virulent viruses. The vCB424 recombinant contained a single nucleotide substitution on an avirulent genetic background, resulting in replacement of Ser-16 of VP4 with Arg-16. Mice infected with vCB424 displayed an intermediate phenotype.     

An RNA stem-loop within the bovine coronavirus nsp1 coding region is a cis-acting element in defective interfering RNA replication

Higher-order cis-acting RNA replication structures have been identified in the 3'- and 5'-terminal untranslated regions (UTRs) of a bovine coronavirus (BCoV) defective interfering (DI) RNA. The UTRs are identical to those in the viral genome, since the 2.2-kb DI RNA is composed of only the two ends of the genome fused between an internal site within the 738-nucleotide (nt) 5'-most coding region (the nsp1, or p28, coding region) and a site just 4 nt upstream of the 3'-most open reading frame (ORF) (the N gene). The joined ends of the viral genome in the DI RNA create a single continuous 1,635-nt ORF, 288 nt of which come from the 738-nt nsp1 coding region. Here, we have analyzed features of the 5'-terminal 288-nt portion of the nsp1 coding region within the continuous ORF that are required for DI RNA replication. We observed that (i) the 5'-terminal 186 nt of the nsp1 coding region are necessary and sufficient for DI RNA replication, (ii) two Mfold-predicted stem-loops within the 186-nt sequence, named SLV (nt 239 to 310) and SLVI (nt 311 to 340), are supported by RNase structure probing and by nucleotide covariation among closely related group 2 coronaviruses, and (iii) SLVI is a required higher-order structure for DI RNA replication based on mutation analyses. The function of SLV has not been evaluated. We conclude that SLVI within the BCoV nsp1 coding region is a higher-order cis-replication element for DI RNA and postulate that it functions similarly in the viral genome.