Due to the requirement of huge amount of starting material, it has been difficult for establishing a ChIP assay for small tissues to detect an association of a protein with transcription factors in vivo. I have recently developed a ChIP assay for transcription factors (Sry and Sox9) that utilizes chromatin from 20 intact embryonic testes (16-18 ts stage) without cross-linking protein to DNA. All ChIP assays published so far utilize cross-linking procedure to retain protein-DNA complex throughout the procedure. Question is “Is it possible to hold your protein-DNA complex for days without perturbing its binding?”.I say yes. My experimental conditions retained that complex. Let’s see if this ChIP assay for transcription factors (Sry and Sox9) can be validated and standardized for several other transcription factors. At the end, there will be a way out for catching up mechanisms underlying early embryonic development.
WSU-UI Center for Reproductive Biology organized a retreat on May 14 and 15 at Orofino, ID. Distinguished Geneticist Dr. Robin Lovell-Badge was invited to deliver a speech on vertebrate sex determination. Dr. Terry Ned who is a renowned reproductive biologist in the nation delivered a Founder's Lecture on how to make a contraceptive to sterilize wild animals. Both speakers gave an elegant speech. Student and Trainee speakers did pretty well. The overall program was successful. At the end of the first day presentation, power went out. Everyone had a candle light dinner. Volleyball game was cancelled, otherwise WSU was said to win the game this year. UI team used to be the bold for ever though.
Presented below is the photograph which reflects the geography of Nepal. It reminded me of my home town.
I participated in 5th
Vertebrate Sex Determination Symposium in Kona, Hawaii from
the 20th through the 24th of April, 2009.
Since I flew from Pullman early in the morning, it was already
11:30 am when plane landed at Kona International Airport.
Another hour was spent for catching a taxi and dropping off my
luggage at the hotel. My previous mentor Dr. Yoshi Nagahama
had already finished his Opening Talk, which I wanted to
attend at any cost. No luck! I also missed the talk of Dr.
Robin Lovell-Badge, who is the godfather of sex determination
research. However, I will be able to hear this talk next month
in Orofino, ID where Dr. Lovell-Badge will give a
guest lecture at the CRB Retreat program of WSU&UI.
Most of the talks were on beta-catenin, Wnt4, FoxL2 and R-
spondin. Non-mammalian talks were equally interesting. The
molecular mechanism of differentiation of fetal and adult
Leydig cells was presented by Dr. Ken Morohashi of Kysushu
University, which was one of the best talks given in this
meeting. My talk was well-received and the ChIP assay I
developed was well-appreciated.
Meeting started at 8:30 and ended at 18:00 everyday except for
22nd afternoon. I took some pictures, which are sitting in the
Picasaweb. Photo
.
The group of Dr. Dagmar Wilhelm at Queensland University have found a gene that could be the potential target for Sry action during sex determination. They immunoprecipitated this fragment from E11.5 dpc gonads using anti-Sry antibody specific to mouse. The approach they have used is novel and there are other potential candidate genes in the list, which they will slowly be characterizing.
The Cerebellin 4 Precursor Gene Is a Direct Target of SRY and SOX9 in Mice Bradford ST, Hiramatsu R, Maddugoda MP, Bernard P, Chaboissier MC, Sinclair A, Schedl A, Harley V, Kanai Y, Koopman P, Wilhelm D. Biol Reprod. 2009 Feb 11. [Epub ahead of print]
In most mammals, the expression of SRY (sex-determining region on the Y chromosome) initiates the development of testes and thus determines the sex of the individual. However, despite the pivotal role of SRY, its mechanism of action remains elusive. One important missing piece of the puzzle is the identification of genes regulated by SRY. In this study we used chromatin immunoprecipitation to identify direct SRY target genes. Anti-mouse SRY antibody precipitated a region 7.5 kb upstream of the transcriptional start site of cerebellin 4 precursor (Cbln4), which encodes a secreted protein. Cbln4 is expressed in Sertoli cells in the developing gonad with a profile mimicking that of the testis-determining gene Sox9 SRY-box containing gene 9). In transgenic XY mouse embryos with reduced Sox9 xpression, Cbln4 expression also was reduced, whereas over-expression of Sox9 in XX mice caused an up-regulation of Cbln4 expression. Finally, ectopic up-regulation of SRY in vivo resulted in ectopic expression of Cbln4. Our findings suggest that both SRY and SOX9 contribute to the male-specific up-regulation of Cbln4 in the developing testis, and identified a direct in vivo target gene of SRY.
Fifth international symposium on the biology of vertebrate sex determination is going to be held at Royal Kona Resort in Kona, Hawaii from April 20 through April 24, 2009. The aim of this symposium is to bring together scientists and students from a wide variety of disciplines with a common interest in sex determination. I am also participating at the meeting and the title is "Neurotrophin-3, a target gene for Sry action". I anticipate meeting with renown scientists in the field. So far, in each symposium, scientists have presented at least one breakthrough in the field. I am anxiously waiting to hear another yet new breakthrough on Sry/Sox9 signalling in the gonad. For details of this and previous meetings, please visit the following website:
I am working in the laboratory of Dr. Michael Skinner, Washington State Univesity. Dr. Skinner is a preeminent figure in reproductive biology and Epigenetics. The finding that the effects of environmental toxic sustances cause epigenetic changes in the germ line that can pass to subsequent generations has received public attention. We are trying to show the world how future diseases can get imprinted in the germ line when life is still within the fetus. Some findings have been ranked one of the top 100 discoveries in the year 2007.
Dr. Skinner keeps his lab running in a very friendly atmosphere. Science is our passion and friendship is our relation.Here is the picture of our team- half the members are absent in the picture, though.
Barack Obama has been elected as the 44th President of United States of America. My heartfelt congratulations to him and the people of United States on this historic victory. His victory confirms that America is the land of opportunities. You aim and work hard, America will fullfil your dreams! Let's hope he will bring about socio-economic changes in the USA and also a peace in the whole world. May he excel in his presidency.
We look forward to getting supports to education and research.
Epigenetic programming of the germ line: effects of endocrine disruptors on the development of transgenerational disease. Anway MD, Skinner MK. Reprod Biomed Online. 2008 Jan;16(1):23-5 School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4231, USA.
Epigenetic programming of the germ line occurs during embryonic development in a sex-specific manner. The male germ line becomes imprinted following sex determination. Environmental influences can alter this epigenetic programming and affect not only the developing offspring, but also potentially subsequent generations. Exposure to an endocrine disruptor (i.e. vinclozolin) during embryonic gonadal sex determination can alter the male germ-line epigenetics (e.g. DNA methylation). The epigenetic mechanism involves the alteration of DNA methylation in the germ line that appears to transmit transgenerational adult onset disease, including spermatogenic defects, prostate disease, kidney disease and cancer.
By Youngson NA, Whitelaw E. Department of Population Studies and Human Genetics, Queensland Institute of Medical Research, Brisbane 4006, Australia; email: emma.whitelaw@qimr.edu.au.
Transgenerational epigenetic effects include all processes that have evolved to achieve the nongenetic determination of phenotype. There has been a long-standing interest in this area from evolutionary biologists, who refer to it as non-Mendelian inheritance. Transgenerational epigenetic effects include both the physiological and behavioral (intellectual) transfer of information across generations. Although in most cases the underlying molecular mechanisms are not understood, modifications of the chromosomes that pass to the next generation through gametes are sometimes involved, which is called transgenerational epigenetic inheritance. There is a trend for those outside the field of molecular biology to assume that most cases of transgenerational epigenetic effects are the result of transgenerational epigenetic inheritance, in part because of a misunderstanding of the terms. Unfortunately, this is likely to be far from the truth.
