During development of the vertebrate lens, the lens epithelium undergoes a final stage of differentiation into lens fibre cells, during which the major lens proteins, the crystallins, are ...synthesised. Lentoids, comprising clusters of lens fibre cells can also be produced by transdifferentiation from certain non-lens tissues, including neural retina and pigmented retinal epithelium. We have isolated an alpha A2-crystallin genomic probe and used it to study the transcription and processing of alpha A2-crystallin mRNA both during lens development and transdifferentiation. We relate these results to earlier measurements in this laboratory of delta-crystallin transcription and alpha- and delta-crystallin protein synthesis, to compare the expression of these two sets of genes. Tissue specific differences in gene expression were found. delta-crystallin mRNA is transcribed before alpha A2-crystallin RNA in the lens, but after it in transdifferentiating neural retina and pigmented epithelia.
During development of the vertebrate lens, the lens epithelium undergoes a final stage of differentiation into lens fibre cells. Lens fibre cells can also be produced by trans‐differentiation from ...certain extralenticular structures, all of which are of different developmental origin from lens, including embryonic neural retina and retinal pigmented epithelium. Delta‐crystallin is the major lens protein in the chick and appears first in development; it is the major product in trans‐differentiated retina of younger embryos. In both normal differentiation and trans‐differentiation an increase of delta‐crystallin coding RNA is detectable in the nucleus of cells prior to their terminal differentiation into lens fibres. The increase in transcription of delta‐crystallin genes accompanying final differentiation of lens fibres, appears to take place slightly in advance of an increase in the capacity to process and transport this mRNA to the cytoplasm.
RNA sequences coding for the most abundant chicken lens protein, δ-crystallin, were detected at very low levels in day old post atch chick lung, heart, kidney and liver, and in 6 day embryo headless ...bodies. The pattern of cytosine methylation within the CCGG sequences of the 6-crystallin genes was also examined and shown to vary in several non-lens tissues, from several stages of development. Embryonic neural retina, which expresses a higher level of δ-crystallin RHA than the above tissues, is no less methylated in the sites studied than the tissues which have no association with the eye, and is actually more heavily methylated than the kidney. Thus no obvious correlation was found between undennethylation and gene expression.
Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain ...tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.
Double-stranded complementary DNA (cDNA) sequences were prepared from day-old chick lens total polysomal RNA and inserted into the unique PstI restriction site of the plasmid pBR322. Colonies ...containing sequences complementary to abundant lens poly(A)-containing RNA sequences were identified by using lens 32P-labelled cDNA. Some of these clones have been characterized as containing delta-crystallin mRNA coding sequences by genomic DNA blot hybridization and RNA blot hybridizations. Hybridization of labelled DNA from such clones to RNA blots detected four size classes of delta-crystallin RNA sequences, although Southern blots indicated that there are probably only two delta-crystallin genes.
Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. ...Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.
An increase in the activity of mitogen-activated protein kinase (MAPK) has been correlated with the progression of prostate cancer to advanced disease in humans. The serine/threonine protein kinase ...p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of MAPK but its role in prostate cancer has not previously been examined. Increasing RSK isoform 2 (RSK2) levels in the human prostate cancer line, LNCaP, enhanced prostate-specific antigen (PSA) expression, an important diagnostic marker for prostate cancer, whereas inhibiting RSK activity using a RSK-specific inhibitor, 3Ac-SL0101, decreased PSA expression. The RSK2 regulation of PSA expression occurred via a mechanism involving both RSK2 kinase activity and its ability to associate with the coactivator, p300. RNA interference of the androgen receptor (AR) showed that the AR was important in the RSK2-mediated increase in PSA expression. RSK levels are higher in approximately 50% of human prostate cancers compared with normal prostate tissue, which suggests that increased RSK levels may participate in the rise in PSA expression that occurs in prostate cancer. Furthermore, 3Ac-SL0101 inhibited proliferation of the LNCaP line and the androgen-independent human prostate cancer line, PC-3. These results suggest that proliferation of some prostate cancer cells is dependent on RSK activity and support the hypothesis that RSK may be an important chemotherapeutic target for prostate cancer.
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