Chromatin, Epigenetics and Human Inherited Diseases
Joel M. Gottesfeld
Professor of Molecular Biology
The Scripps Research Institute
About the Lecture
It has become clear in recent years that epigenetic modifications are important not only in regulating gene expression within cells; but, also as non-DNA based agents of inheritance that can cause genetically heritable diseases. One important type of epigentic modification that can alter gene expression in human cells is postsynthetic modification of histones proteins. These proteins have an essential role in packaging DNA into chromosomes and they undergo a remarkable variety of post-translational modifications that affect their interaction with DNA and with other proteins involved in regulating gene expression, DNA replication, and folding and packing DNA into higher order structures. We have found that alterations in these modifications underlie a heritable disease.
Friedreich’s ataxia (FRDA) is a crippling degenerative nerve disease that also leads to diabetes and heart disease. Patients typically die in early adulthood from heart failure. We have found that FRDA is caused by a heritable mutation that results in a reduction in histone acetylation, which silences the frataxin gene. We have developed small molecules that inhibit the enzyme histone deacetylase and have been able to show that these molecules increase acetylation of the histones associated with DNA (in the form of chromatin), and that they reactivate frataxin expression in models of FRDA. The results suggest that epigenetic alterations that cause disease can be remediated by appropriately targeting small molecules to enzymes that mediate epigenetic marks.
FRDA is one of a class of diseases referred to as triplet repeat diseases, because they are characterized by an expansion of a simple triplet repeat in particular gene-associated regions of DNA. Huntington’s disease is also a triplet repeat disease and we are developing similar classes of compounds as therapeutics for this disease as well.
This lecture will address epigenetics generally, the role of epigenetic alteration in Friedreich’s ataxia and other epigenetic diseases, the use of epigenetic targets for therapeutic development and our progress in developing promising new molecules for treating these diseases.
About the Speaker
JOEL M. GOTTESFELD is Professor of Molecular Biology at The Scripps Research Institute in La Jolla, California, and he works with Repligen Corporation in developing novel compounds from his laboratory that target epigenetic diseases. Joel earned a B.S. at UC-Berkeley, an M.S. at Oxford University as a Fulbright Fellow, and a Ph.D. at the California Institute of Technology. He did postdoctoral work at the MRC Laboratory of Molecular Biology in Cambridge, England as a Helen Hay Whitney Fellow. Thereafter he joined the faculty at the Scripps Research Institute, where he is a Professor today. Joel also served for two years as Division Head at the Medical Biology Institute in La Jolla. He has published well over 100 research and technical papers. He has been thesis advisor to numerous doctoral students and has hosted more than 25 postdoctoral fellows in his lab. He has been co-chair of Gordon Research Conferences on Gene Regulation and Chromatin Structure, and for many years was a principle organizer of the Asilomar Chromatin Conference. He has served as peer reviewer for many journals, served on numerous NIH peer review panels, and currently is Associate Editor and a member of the Editorial Board of the Journal of Biological Chemistry.
President Robin Taylor called the 2,273rd meeting to order at 8:19 pm October 15, 2010 in the Powell Auditorium of the Cosmos Club. Ms. Taylor introduced one new member of the Society. She announce the deaths of two members. The minutes of the 2,272nd meeting were read and approved.
Ms. Taylor then introduced the speaker of the evening, Mr. Joel M. Gottesfeld of The Scripps Research Institute. Mr. Gottesfeld spoke on “Chromatin, Epigenetics, and Human Inherited Diseases.”
Mr. Gottesfeld began by defining epigenetics: the study of inherited changes in phenotype caused by changes in gene expression that are not due to underlying DNA sequence.
Each cell type expresses a set of genes. With minor exceptions, all cells have the same DNA. It is the activation of genes that determines the cell type – neuron, muscle, blood, and so on.
Two mechanisms account for critical effect of activation. One is postsynthetic modification of histones by addition of a small chemical group. Histones are the proteins that package DNA in the cell nucleii. The other is DNA methylation, or modification of cytosine residues in DNA that alter simple methyl to yield 5'-methylcytosine.
Mr. Gottesfeld spoke mostly of eucaryotes, which have their DNA in nuclei. Procaryotes have no nucleii.
Diseases in eucaryotes can be caused by anomalies in gene activation. One such disease is Friedreich’s Ataxia.
Gene expression is regulated by transcription, RNA processing, mRNA export from the nucleus, and translation. Translation, the copying of DNA into RNA, is the means of most interest here.
This process is mediated by an enzyme, RNA polymerase II. It works with a set of proteins called transcription factors, some of which recognize particular DNA sequences, the promoter sequence of a gene. These DNA sequences recruit accessory proteins, which in turn recruit RNA polymerase to genes.
DNA in a nucleus is packaged into chromatin. There are many layers of chromatin structure that influence gene expression.
A nucleosome is a combination of DNA with proteins called histones. DNA is on the outside, histones are on the inside. There are four core histones – H3, H4, H2A, and H2B. The middle of this structure is compacted, and there are “tails,” or extensions, sticking out in many places. It is in the tail part of the structure that the reversible modifications occur.
What do histone modifications do? They are a code for gene expression. They write the histone codes which are read by proteins that recognize modified histones. They recruit positive and negative regulatory factors. Histone acetylation affects chromatin structure.
But it’s not that simple: small RNAs, such as microRNAs, may also be involved in establishing heterochromatin, through an RNA transcriptional silencing process. This may hybridize RNA transcripts. Also, DNA methylation affects gene expression through a protein which recruits enzymes that modify chromatin proteins.
So epigenetics has a role in developmental programs, cellular differentiation, genetic imprinting, and X-chromosome inactivation. But it is also involved in disease, including various human cancers, Rett syndrome, and triplet repeat disorders. Both fragile X Syndrome and Friedreich’s ataxia are triplet repeat disorders, sometimes called trinucleotide expansion repeat disorders. More than 20 neurological disorders are caused by this simple chemical process.
Friedreich’s ataxia is caused by a mutation in a gene called frataxin. It encodes protein involved in storage of iron in cells. Triplets of GAA repeats are expanded in a non-coding region of the gene. Long GAA repeats lead to a block of RNA polymerase elongation.
It is an autosomal recessive disease. About 1 in 40,000 individuals, among Caucasians, is affected. Earlier onset and more severe symptoms are associated with longer repeats. Symptoms result from degeneration of the central and peripheral nervous systems. Death usually arrives in early adulthood. No effective treatments have been available.
Mr. Gottesfeld guessed that gene activation would be therapeutic, since the coding potential of the frataxin gene is unaffected. He looked for answers to the question, how do the repeats cause the gene to be silenced? He found it is caused by heterochromatin, or condensed inactive chromatin. Histones on the FSN gene chromatin are hypoacetylated and one of the histones is hypermethylated. These conditions combine to cause chromatin condensation and frataxin gene silencing.
What can be done to reverse heterochromatin and frataxin gene silencing? There are a number of histone deacetylase inhibitors (HDACi). A series of tests was run in FRDA cell lines and in primary lymphocytes from patients.
There is a mouse model for Friedreich’s ataxia maintained at the Pandolfo Laboratory in Brussels. Treatment of these animals with one of the HDACi compounds, called Compound 106, yielded promising results. It crosses the blood-brain barrier and restores brain, cerebellar and heart frataxin and mRNA levels to normal. Mice treated with the compound also were better able to perch on a freely spinning bar called a Rotarod. This is apparently a great achievement for an ataxic mouse.
Will it work for humans? There is hope. Although Mr. Gottesfeld came to the end of the road in his lab, Repligen Corporation has licensed the HDAC inhibitors for development. There is much work to be done. The compounds will be tested for pharmacokinetics, cell permeability, metabolic stability, receptor cross-reactivity, cytotoxic properties, and other factors. They’ve synthesized 200 derivatives, 61 of them active ones. One of them, compound 109, looks better than compound 106 and has been chosen for pre-clinical development. Safety trials in animals have been completed. Early safety trials in human subjects are planned.
Mr. Gottesfeld is continuing laboratory work on related chemicals and their effects on cells. He has developed a human neuronal cell to use as a disease model. They take skin cells from FRDA patients and transduce them and turn them into pluripotent cells. They then turn those pluripotent cells into neurons. These neurons respond to compound 109 positively.
To sum up, the importance of epigenetics is clear. It is essential for normal development, and if it goes awry, it causes horrible diseases such as Friedreich’s ataxia. It is a disease of chromatin and epigenetics. The triplet repeat GAA induces heterochromatin and silences the gene. Studies to date indicate that it will be possible to develop chemicals that will treat the problem.
The first question was about whether he had used software to come up with the derivatives. He did not. This class of molecules are well studied; they know what features of the molecule do what. They just made derivatives and tested them.
Next, someone asked, since the disease is also associated with heart disease, is it possible that smaller numbers of the GAA repeats cause heart disease even though they are insufficient to cause Freidriech’s ataxia? We don’t know yet, Mr. Gottesfeld said. That will be tested in later clinical trials. The animal models for the disease have been poor, and have not shed any light on a possible heart disease connection.
Another person asked about the “hot button,” stem cells. Mr. Gottesfeld reiterated that they don’t use stem cells. They transform skin cells into pluripotent cells, which are much like stem cells, but they are not.
Another question was about HDAC inhibitors and other diseases, perhaps involving other kinds of gene silencing. Mr. Gottesfeld indicated there is a large literature on Huntington’s disease, a polyglutamine expansion disorder. The Huntington protein sequesters histone acetyl transferase. His laboratory tested compound 106 in a Huntington’s mouse model and saw the same kind of benefits as for Freidriech’s ataxia. As a result, he said, they received a very nice grant from NIH to investigate these molecules effects on Huntington’s.
After the talk, Ms. Taylor presented a plaque commemorating the occasion. She made the usual housekeeping announcements. She invited visitors to apply for membership. She announced the 2,274th meeting. Finally, at 9:51 pm, she adjourned the 2,273rd meeting to the social hour.
The weather: Unremarkable
The temperature: 13°C
Ronald O. Hietala,