The group at UCSF Benioff Children's Hospital is researching epigenetic mechanisms involved in gene expression reprogramming. They have two main projects: 1) studying AID, an enzyme that can demethylate DNA and may play a role in the epithelial-mesenchymal transition (EMT) process linked to cancer metastasis, and 2) characterizing super-enhancers (SEs), clusters of enhancers that regulate genes key to cell identity and cancer, to identify new therapeutic targets. They are also developing a CRISPR-based gene therapy for hemoglobinopathies like sickle cell disease.

1. My group at UCSF Benioff Children's Hospital and Research Institute at Oakland,
is interested in the epigenetic mechanisms involved in reprogramming of gene
expression. We are also interested in developing gene therapies for genetic
diseases. We currently have two main research projects in epigenetics, one
regarding Activation-Induced Cytidine Deaminase (AID), a novel DNA
demethylase, and another one focusing on Super-Enhancers (SEs), also known
as stretch enhancers, novel regulatory elements important for cell identity and
oncogenesis.
Epigenetics
AID research AID is an enzyme well known for its role in immunoglobulin
diversification. Canonical AID functions in B cells are class switch recombination,
somatic hypermutation and gene conversion. More recent studies have
described AID as being expressed during early development in various
organisms and in pluripotent cells in mammals. AID’s functions in B lymphocytes
could not explain AID’s role in non-immune cells. This led to the finding that AID
is able to demethylate DNA in a variety of epigenetic reprogramming phenomena
in vertebrates. Since AID’s only activity is cytosine deamination, DNA
demethylation is achieved indirectly by deamination of methylcytosine/cytosine,
creating mismatches (T:G/U:G) that are repaired by ubiquitous DNA repair
factors.
The evidence that AID mediates cytosine demethylation in epigenetic
reprogramming events, the resemblance of the epithelial-mesenchymal transition
(EMT) to reprogramming, and changes in methylation during the EMT, suggested
to us that AID might have a role in the EMT.
The EMT is the process by which epithelial cells acquire mobile and invasive
properties, and is the limiting step in the initiation of metastases. It is elicited by
inflammatory signals contributed by the tumor microenvironment and infiltrating
lymphocytes. We have found that AID is necessary for mammary epithelial cells
to go through the EMT. We showed that AID is induced by inflammatory signals
that concomitantly induce the EMT, and that AID facilitates demethylation of
promoters of genes that are key players of the EMT. Therefore insights into the
mechanism by which AID demethylates DNA, controls gene expression and
facilitates the EMT, are crucial to find inhibitors that will inactivate this pathway.
DNA demethylation is a novel function of AID, and given AID expression in
pluripotent and germ cells and the phylogenetic distribution of AID and
immunoglobulin diversification, we think that DNA demethylation may be its
primordial function. We are currently carrying out “genomewide” approaches:
ChIP-Seq, RNA-Seq and Methyl-Seq to find the direct targets of AID in luminal
breast cancer cells during the EMT.
We have also found that AID is expressed in human breast tumor specimens,
and we have established a mouse model of breast carcinogenesis that
recapitulates most steps of the human disease, to study the consequences of
AID expression, or lack of, during cancer progression.
AID is also pathologically expressed in various epithelial cells/tissues under
chronic inflammatory conditions that lead to cancer development, such as

2. ulcerative cholitis, Crohn’s disease and infections with Hepatitis C virus and
Helicobacter pylori. We have found that AID is induced upon NOD1 signaling in
colon cancer cells, and that AID is necessary for the expression of the
inflammatory cytokine interleukin-6 (IL6), under the same stimulus. IL6 secretion
generates a positive feedback loop that maintains inflammation, AID expression
and promotes tumorigenesis, therefore implicating AID as a cancer progression
factor, once inflammation is initiated. We are currently using mouse models (wild
type and AID KO mice) to establish the mechanism/s of AID induction under
chronic inflammatory conditions and its consequences in the pathophysiology of
ulcerative cholitis and Crohn’s diseases.
Super-enhancer research
Super-enhancers (SEs) are clusters of transcriptional enhancers that function as
units to regulate expression of genes that are key for cell identity, survival, and
oncogenesis. They have recently been identified in multiple human cell types and
cancers, and are particularly enriched in acetylated H3K27 and chromatin
binding factors such as the Mediator complex. Disruption of SEs impairs cancer
cell survival. Expression of genes that are regulated by SEs is extremely
sensitive to mild variations in the amount and/or composition of the transcription
factors and cofactors bound to the SEs. These genes usually encode key
regulators of tumor cell state. SEs are bound by transcriptional activators, but
they differ from canonical enhancers in size, transcription factor density and
content, ability to activate transcription, and perhaps most importantly, sensitivity
to perturbation.
Characterization of SEs has proven to be an extremely useful tool to find new
transcriptional regulatory nodes, and producing several oncogene candidates to
be considered as therapeutic targets.
We are characterizing SEs in a variety of child and adult tumors by ChIP-Seq
and characterizing the factors necessary for their de novo formation by en-ChIP.
The purpose of cataloguing SEs is to identify genes, transcription factors, and
regulatory elements important for carcinogenesis and progression. These
comparative and comprehensive studies may uncover new regulatory circuits
that are characteristic of specific cancers in general, or of specific cancer
subtypes.
We have improved existent algorithms for SE calling and used them to identify
several SEs in breast cancer cells that regulate the expression of new factors
that may have a role in breast cancer. We are testing the function of these SEs
by a novel strategy using the recently developed CRISPR system that permits
targeted disruption of chromatin function.
We are also using the epigenetic datasets generated through these studies and
publicly available ones to perform integrated bioinformatic analysis for better
stratification of various cancers and identification of tissue of origin.

3. Gene Therapy
Hemoglobinopathies
Another area of interest is the development of a gene therapy for the treatment of
hemoglobinopathies such as sickle cell disease and some -thalassemias. We
are using the CRISPR system to deliver genetic modifications into hematopoietic
stem cells (HSCs) that once transplanted are able to reconstitute the immune
system of the patient. Since this therapy involves an autologous transplant there
is no need to look for matching donors, and poses no complications from graft
rejection. We are designing and testing various strategies to maximize the
correction rate in HSCs.