Research | iwase-lab

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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

Disrupted intricacy of histone H3K4 methylation in neurodevelopmental disorders. Vallianatos CN, and Iwase S. Epigenomics, 2015. (Review article)
Yin-yang actions of histone methylation regulatory complexes in the brain. Garay PM, Wallner MA, Iwase S. Epigenomics. 2016. (Review article)
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
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
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
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
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
RAI1 Regulates Activity-Dependent Nascent Transcription and Synaptic Scaling. Garay PM, Chen A, Tsukahara T, Rodríguez Díaz JC, Kohen R, Althaus JC, Wallner MA, Giger RJ, Jones KS, Sutton MA, Iwase S. Cell Rep 2020.
LSD1/KDM1A Maintains Genome-wide Homeostasis of Transcriptional Enhancers. Agarwal S, Garay PM, Nakamura Y. Macfarlan T, Ren B, and Iwase S. Genome Res 2021.

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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

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)
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
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

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Soma-to-germline transformation in neurodevelopmental disorders?

An underappreciated observation in models of neurodevelopmental disorders (NDD) is that several display ectopic expression of germline genes, which should only be expressed in the testis or ovary. These NDD-associated chromatin regulators normally condense chromatin structure and silence transcription. Therefore, a likely explanation is that the germline genes are de-repressed due to impaired transcriptional silencing. We seek to address the question—Does ectopic germline gene expression contribute to NDDs?

Our focus is lysine demethylase 5c (KDM5C), whose loss of function is responsible for an NDD characterized by intellectual disability, autistic features, and aggressive behavior. KDM5C reverses H3K4me, a hallmark of transcriptionally-engaged chromatin, and the modification is conserved from yeast to humans. Kdm5c-knockout (KO) mouse model recapitulates the key features of the behaviors seen in human patients. In addition, our unbiased survey of gene misregulation with RNA-seq revealed that the top altered genes were germline-specific genes, derepressed in Kdm5c-KO brain tissues. We hypothesize that germline gene expression bestows ‘germcellness’ to the mutant brain and contributes to neurological complications of Kdm5c-KO mice. This project will use cell biological and functional genomics approaches to test if Kdm5c-KO mouse brains show germline phenotypes such as genomic imprinting erasure.

This project addresses the impact of non-brain genes on neurodevelopmental defects for the first time. It illuminates germline gene silencing and genomic imprinting as a new mechanism underlying KDM5C disorders. Loss of functions of three other NDD genes, DNMT3B, EHMT1, and MECP2, leads to germline gene derepression in the brain. Thus, ectopic germline gene expression is relevant to a subset NDDs beyond KDM5C disorder. The clear distinction between somatic cells and germ cells has been proposed to be a driver of multicellularity, which emerged approximately 1.5 billion years ago. Silencing of germline genes in somatic cells might have enabled this evolutionary innovation. Thus, our work lays a foundation for turning a deep evolutionary root underlying NDDs into a therapeutic strategy.

Publications on Germline Gene Repression

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
Soma-to-germline transformation in chromatin-linked neurodevelopmental disorders? Bonefas K and Iwase S. FEBS J 2022.

Identifying the molecular function of orphan chromatin factors

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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.

We do not know the molecular function of some chromatin factors whose mutations are responsible for neurodevelopmental disorders.

For example, ATRX is mutated in Alpha-thalassemia mental retardation, X-linked (ATR-X) syndrome. 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.

In addition, RAI1 mutations cause Smith-Magenis Syndrome and encodes a nucleosome-binding protein with an ADD domain. However, we do not know if RAI1 recognizes specific histone or DNA modification. We are exploring the roles of RAI1 using chromatin biochemistry approaches.

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

Publications on Orphan Chromatin Factors

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

Mechanistic link between virus, chromatin, and neurodevelopmental disorders

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Genetic studies of neurodevelopmental disorders (NDDs) have revealed that mutations in chromatin regulators are a major cause of autism spectrum disorders, intellectual disability, and schizophrenia. However, how chromatin dysregulation leads to cognitive deficits remain elusive. As a mechanism, most research has focused on cellular gene regulation underlying neurodevelopment by the mutated chromatin regulators.

Meanwhile, common viruses are chronically present in a significant fraction of populations. For example, > 60% of people have cytomegalovirus (CMV) and herpes simplex virus (HSVs) in their immune cells or neurons, yet their presence is believed to be inconsequential unless individuals are immunocompromised. We accidentally observed that the CMV promoter used in reporter assays was strongly suppressed by PHF21A, a histone-binding protein whose mutation is responsible for a rare NDD called Potocki-Shafer Syndrome. PHF21A is a reader protein for unmethylated H3K4 (H3K4me0), which is generated by LSD1-medaited histone demethylation. Virologists have found that PHF21A-associated factors regulate the latency of CMV and HSV. These observations made us realize that the common persistent virus can modify or even mediate the cognitive deficits caused by chromatin factor mutations.

In this new project, we aim to understand how virus infection modulates chromatin-linked neurodevelopmental disorder phenotype outcomes. Upon infection, herpesviruses acquire histone-associated proteins to exist in an episomal or latency state where chromatin regulators can alter viral gene expression by modifying histone marks at the promoter region of these viruses. We are exploring the roles of PHF21A in these viral processes. This research may unveil the common persistent virus as a key contributor to neurodevelopmental disorders caused by chromatin dysregulation. Herpesviruses have been infecting and co-diverging with their vertebrate hosts for hundreds of millions of years, and curiously, the emergence of herpesvirus, PHF21A, and vertebrate coincides. Thus, the research will shed light on how the tug-of-war with viruses shaped normal and pathological human brains.

Tool Development for Chromatin Neurobiology

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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 a 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 harbor 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.

We also applied nascent RNA sequencing in neurons to precisely monitor activity-dependent transcription, rather than steady-state mRNA levels. This led to the identification of a new role of RAI1 suppressing transcriptional programs invoked by chronic neuronal inactivation.

These methods will help us to decipher the roles of chromatin regulators in the brain and the molecular mechanisms underlying neurodevelopmental disorders. For example, with the DLAF method, we are characterizing promoter usage in neurons undergoing neural network remodeling.

Publications on Chromatin Neurobiology Tool Development

Sequencing of first-strand cDNA library reveals full-length transcriptomes. Agarwal S, Macfarlan TS, Sartor MA, Iwase S. Nature communications, 2015;6:6002.
RAI1 Regulates Activity-Dependent Nascent Transcription and Synaptic Scaling. Garay PM, Chen A, Tsukahara T, Rodríguez Díaz JC, Kohen R, Althaus JC, Wallner MA, Giger RJ, Jones KS, Sutton MA, Iwase S.