©2018 by Chromatin Neurobiology Lab. 

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The University of Michigan Medical School

Human Genetics Department

1

Histone H3K4 Methylopathies of the Brain

Methylation of histone H3K4 (H3K4me) is one of the most extensively-regulated post-transnational modifications in our cells. H3K4me plays defined roles in distinct steps of transcriptional regulations. 6 enzymes are known to 'write' H3K4me, while 6 enzymes can 'erase' this mark in our cells. Of the 12 enzymes, strikingly 8 of them have been genetically associated with neurodevelopmental disorders. In addition three 'readers' of H3K4me are responsible for specific neurodevelopmental conditions. These genetics observations tell us that intricate regulation of H3K4 methylation is critical for normal brain development. However, the roles of each H3K4me regulators in brain development and function remain largely unknown.

 

We have previously identified that KDM5C (aka SMCX or JARID1C), one of the most frequently mutated genes in intellectual disability syndromes, encodes an enzyme that removes a methyl group (an ‘eraser’ enzyme) from histone H3 at the lysine 4 residue (H3K4me). Our analyses were the first to link the dynamic nature of histone methylation with human cognitive development/function. 

More recently, we have found novel roles of RAI1, Smith-Magenis Syndrome gene (SMS) gene, in specific transcriptional program that is driven by reduced network activity. In collaboration with Sutton laboratory at the University of Michigan, we also discovered that the roles of RAI1 in transcription underlie homeostatic synaptic plasticity. These research provide mechanistic understanding of SMS and forms a ground for future therapeutic intervention. 

Our research priorities is to better understand the interrelationship between these K4me regulators and whether they target the same set of genes or distinct regulatory regions. We also wish to determine the cellular/behavioral consequences of the loss of these factors. To address these questions, we will employ a variety of approaches including mouse genetics, cytological/histological examinations, the biochemical characterization of mutations, and genome-wide expression/mapping studies. We also aim to establish links between specific molecules and animal behaviors by collaborating with experts in electrophysiology and mouse behavioral studies.

Publications on Brain H3K4 Methylopathies

  1. Disrupted intricacy of histone H3K4 methylation in neurodevelopmental disorders. Vallianatos CN, and Iwase S. Epigenomics, 2015. (Review article)

  2. Yin-yang actions of histone methylation regulatory complexes in the brain. Garay PM, Wallner MA, Iwase S. Epigenomics. 2016. (Review article)

  3. The X-Linked Mental Retardation Gene SMCX/JARID1C Defines a Family of Histone H3 Lysine 4 Demethylases.                                      Iwase S, Lan F, Bayliss P, de la Torre-Ubieta L, Huarte M, Qi HH, Whetstine JR, Bonni A, Roberts TM, Shi Y. Cell 2007;128(6):1077-1088

  4. A Mouse Model of X-linked Intellectual Disability Associated with Impaired Removal of Histone Methylation.
    Iwase S*, Brookes E, Agarwal S, Badeaux AI, Ito H, Vallianatos CN, Tomassy GS, Kasza T, Lin G, Thompson A, Gu L, Kwan KY, Chen C, Sartor MA, Egan B, Xu J*, Shi Y* *Co-corresponding authors. Cell Reports 2016;14(5):1000-9

  5. Loss of Kdm5c Causes Spurious Transcription and Prevents the Fine-Tuning of Activity-Regulated Enhancers in Neurons.
    Scandaglia M, Lopez-Atalaya JP, Medrano-Fernandez A, Lopez-Cascales MT, Del Blanco B, Lipinski M, Benito E, Olivares R, Iwase S, Shi Y, Barco A. Cell reports 2017;21(1):47-59

  6. Transcriptome analysis revealed impaired cAMP responsiveness in PHF21A-deficient human cells.
    Porter RS, Murata-Nakamura Y, Nagasu H, Kim HG, Iwase S. Neuroscience 2017;17:30365-2

  7. Altered Gene-Regulatory Function of KDM5C by a Novel Mutation Associated With Autism and Intellectual Disability.
    Vallianatos CN, Farrehi C, Friez MJ, Burmeister M, Keegan CE, Iwase S. Front Mol Neurosci 2018;11:104

  8. LSD1/KDM1A Maintains Genome-wide Homeostasis of Transcriptional Enhancers
    Agarwal S, Garay PM, Nakamura Y. Macfarlan T, Ren B, and Iwase S. BioRxiv. 

  9. RAI1 Regulates Activity-Dependent Nascent Transcription and Synaptic Scaling
    Patricia M. Garay, Alex Chen, Takao Tsukahara, Rafi Kohen, J. Christian Althaus, Margarete A. Wallner, Roman J. Giger, Michael A. Sutton & Shigeki Iwase, BioRxiv. 

2

Is Neuronal Chromatin Special? 

It is widely accepted that cell-type-specific gene expression is primarily achieved by cell-type-specific presence of transcription factors (TFs), which bind to cognate DNA sequences. TFs then initiate changes in higher-order chromatin structures by recruiting chromatin modifiers, including histone-modifying enzymes. Unlike TFs, chromatin modifiers tend to be ubiquitously expressed. It is unknown whether histone modifiers act in a cell-type-specific manner. Whereas dozens of chemical marks can decorate histones, regulators of histone methyl-ation are more frequently found to be mutated in neurodevelopmental disorders (NDDs) such as intellectual disabilities (IDs). Why is the brain so sensitive to dysregulation of histone methylation? Is methyl-histone regula-tion in neurons unique compared to other cell types? Investigation of a limited number of cell types, cancer-cell lines, and embryonic stem cells has hampered our ability to address these questions.  

The overarching goal of our research group is to contribute to the understanding of how methyl-histone regulations underlie normal and pathological brain functions. A recent study discovered that 3–27 nucleotide “micro-exons” are predominantly generated in neurons via alternative splicing. Microexons are evolutionarily con-served, misregulated in NDD patients, and predicted to influence protein–protein interactions. We mined the published data and found that more than 70 chromatin regulators contain neuron-specific microexons, implying that microexons are a driving force in the generation of neuron-specific chromatin landscapes.  

Publications on Neuron-specific Chromatin Regulation

  1. Neuron-specific alternative splicing of transcriptional machineries: Implications for neurodevelopmental disorders.                                      Porter RS, Jaamour F, Iwase S. Mol Cell Neurosci. 2018 Mar;87:35-45. (Review article)

  2. A component of BRAF-HDAC complex, BHC80, is required for neonatal survival in mice
    Iwase S, Shono N, Honda A, Nakanishi T, Kashiwabara S, Takahashi S, Baba T. FEBS Lett. 2006;580(13):3129-3135

  3. Characterization of BHC80 in BRAF-HDAC complex, involved in neuron-specific gene repression
    Iwase S, Januma A, Miyamoto K, Shono N, Honda A, Yanagisawa J, Baba T. Biochem. Biophys. Res. Commun. 2004;322(2):601-608

3

Heterochromatin and the Brain

Constitutive heterochromatin attracts less attention than other chromatin types because it does not contain genes. Heterochromatin is also packed more tightly, and thus tends to be transcriptionally silent. However, it appears that a portion of the constitutive heterochromatin plays a pivotal role in chromosome segregation. Faithful chromosome separation is particularly important for the adequate supply of neuronal stem cells during development as well as in adulthood. Moreover, the prevention of ectopic heterochromatin formation appears to be an important gene regulatory system as it can cause mis-silencing of genes. The proper control of heterochromatin is a requirement for maintaining appropriate gene expression in mature neurons as in other cells. However, very limited knowledge is currently available on the control of heterochromatin and the consequences of this for brain function. 

One of the heterochromatin components found to be mutated in ID is ATRX (Alpha-thalassemia mental retardation, X-linked). Patients with ATR-X syndrome suffer from a wide range of developmental abnormalities including IDs. Mutations are predominantly found in two regions of the ATRX protein: a cysteine-rich-domain, known as ADD, and an enzymatic domain, which aids the incorporation of H3.3, a variant of histone H3, into DNA. We have previously identified that the ADD domain is a “reader” module for methylated lysine 9 of histone H3 (H3K9me), and that the interaction between ADD and H3K9me is crucial for the recruitment of ATRX onto heterochromatin. 

An open question at present is why ATRX needs to be tethered to heterochromatin and then deposit H3.3 i.e. how does this contribute to the faithful DNA separation of neuronal stem cells and gene regulation in post-mitotic neurons? To address this, we will employ both biochemical and cellular approaches. These experiments will likely provide significant new insights into the poorly understood link between heterochromatin and IDs, and may provide important clues to the development of a future therapeutic intervention for these disorders. 

Publications on Brain Heterochromatin

  1. ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome
    Iwase S, Xiang B, Ghosh S, Ren T, Lewis PW, Cochrane JC, Allis CD, Picketts DJ, Patel DJ, Li H, Shi Y
    Nat. Struct. Mol. Biol.2011;18(7):769-776

Tool Development for Chromatin Neurobiology

4

New tools allow us to observe what we have not seen before. We strive to develop new tools for chromatin neurobiology study. For example, we developed new RNAseq library generation method.  

Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 50 and 30 ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 50 and 30 ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 50 ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the ‘full-length’ transcriptome with a single library.

This method will help us to decipher the roles of chromatin regulators in the brain and the molecular mechanisms underlying neurodevelopmental disorders.

Publications on Chromatin Neurobiology Tool Development

  1. Sequencing of first-strand cDNA library reveals full-length transcriptomes.
    Agarwal S, Macfarlan TS, Sartor MA, Iwase S. Nature communications, 2015;6:6002