whitehead lecture 2/3/2020 © dj waxman 1wi.mit.edu/files/wi/cfile/whitehead_sexdiffs...

Whitehead lecture 2/3/2020 © DJ Waxman 1

David J. Waxman

Department of Biology and Bioinformatics ProgramDept. of Medicine, Dept. of Biomedical Engineering

Boston [email protected]

Whitehead InstituteSex Differences in Health and Disease Seminar Series

February 3, 2020

Sex Differences in the Liver:Metabolism, liver disease and epigenetic

mechanisms

Liver is a Sexually Dimorphic Organ

Yokoyama et al , 2005

Liver fibrosisand cirrhosis

Liver Cancer

Alcoholicliver injury

Autoimmune hepatitis

At the disease level:

Ø Humans show significant sex differences in pharmacokinetics [how the body processes a drug], and pharmacodynamics [how the body responds to a drug], which may impact drug efficacy and safety

Ø This in part reflects sex differences in liver metabolism by both phase I and phase II drug-metabolizing enzymes

Human sex differences in drug metabolism

More rapid metabolism

in males

More rapid metabolism in females


Liver is a Sexually Dimorphic Organ

• Sex differences in levels of enzymes of liver drug metabolism:P450 metabolism enzymes, drug conjugation enzymes, transporter proteins

At the enzyme (protein) level:

• Sex-differences in levels of enzymes of hepatic steroid and lipid metabolism, contributing to male-biased cardiovascular disease risk

https://translate.bio/rna-therapeutics/

Sex differences in the rate at which certain genes are

transcribed to make their mRNAs

At the mRNA and gene expression level:[how actively a gene is transcribed to make its mRNA]

Sex-differencein rate

The bipotential gonad is directed towards testis development by the SRY gene on the Y chromosome. Testosterone production begins during fetal life, surges at birth, then remains low until puberty. In females, the ovary develops due to the lack of SRY and remains quiescent until puberty, when cyclical estradiol production begins. Genes on the X and Y chromosome are capable of directly controlling sex differences.

Sources of Sex Differences: Hormones vs Chromosomes

Demarest & McCarthy (2015) J Bioenerg Biomembr

Sex Chromosomes

Sex diffs in hypothalamo-pituitary activity at puberty and adulthood

XY

XX Incomplete inactivation

of X-chr genes

Expression ofY-chr genes Direct Chromosomal

Effects onSex Differences

Sex Steroids

1

23 Pituitary

GrowthHormonesecretion

Is it androgen and estrogen? Mostly not (at least, not directly). Only 4% of sex-biased genes are direct functional targets of androgen or estrogen receptors in mouse liver [D Zheng et al, Mol Cell Endo 2018].

Is it chromosomal sex? No, we can masculinize a female liver, and can feminize a male liver, by pituitary hormone administration in animal models.

Is it the sex-specific pattern of pituitary GH secretion? Yes!

What regulates liver sex differences?


HypothalamusSOMATOSTATIN GHRH

+

IGF-1

GH Signaling by Liver

Growth and Metabolic EffectsDirect and indirect, some sex-dependent

Pulsatile GHStimulation

HYPOTHALAMO-PITUITARY GH AXIS

Pituitary GH (191 AAs)

production and secretion

-

-+

Neonatal Testosterone programs the male hypothalamus to regulate pituitary

GH release patterns at puberty

Persistent GHStimulation

M F

Sex-specific temporal plasma GH patternsPituitary GH secretion is sex-specific in rats, mice and humans

Waxman DJ et al, PNAS 1991Adams JM et al, Endocrinol 2014Jaffe CA et al, JCI 1998

156

Jaffe et al.

volution-estimated GH secretory patterns are plotted in thebottom panel and the GH concentrations derived from thesesecretion estimates are overlaid on the measured concentra-tion profiles. The mean (

6

SE) GH concentrations for theeight men and eight women are given in Fig. 2. The 24-h pro-files in men were characterized by the presence of a dominantnocturnal pulse with much smaller pulses at other times of theday. In contrast, GH secretion in women was more continuous,with pulses of similar amplitude throughout the 24 h.

Several methods were used to quantify the differences inthe pattern of GH secretion in men and women (Table I). As ameasure of uniformity of pulse amplitude, a SD for each sub-ject’s GH pulse amplitudes during the baseline study was cal-culated. GH pulse amplitudes were logarithmically trans-formed before analysis. This estimate of variation was greaterin men than in women (7.98 vs. 5.31

m

g/liter;

P

5

0.001). Asimilar analysis was performed after logarithmic transforma-tion of the 145 daily GH concentrations. A higher SD for daily

GH was found in men (5.22 vs. 4.03

m

g/liter;

P

5

0.04). Fig. 3shows a histogram of GH concentrations in these two groups.GH concentrations were either equal to or above the assay de-tection limit 98% of the time in men and 100% of the time inwomen. Although there was no difference between men andwomen in terms of absolute GH nadir, there was a differencein the frequency of biologically low GH concentrations. Reu-tens et al. (22) recently reported that subjects with severe or-ganic GH deficiency had virtually all plasma GH concentra-tions

,

0.5

m

g/liter during 24-h sampling. Using this value asan estimate of the minimum GH concentration for bioactivity,only 35

6

4% of the spontaneous plasma GH measurements inmen were above this value, whereas 53

6

6% of the concentra-tions in women exceeded this limit (

P

5

0.04).Deconvolution was used to determine differences in GH

secretion that would account for gender-specific GH concen-tration profiles. Fig. 4 (

top

) shows the BPF for men andwomen calculated as the deconvolution estimated pulse fre-

Figure 2. Composite picture of plasma GH con-centration profiles (mean6SE) in eight men (top) and eight women (bottom) during saline infusions.

156

Jaffe et al.

volution-estimated GH secretory patterns are plotted in thebottom panel and the GH concentrations derived from thesesecretion estimates are overlaid on the measured concentra-tion profiles. The mean (

6

SE) GH concentrations for theeight men and eight women are given in Fig. 2. The 24-h pro-files in men were characterized by the presence of a dominantnocturnal pulse with much smaller pulses at other times of theday. In contrast, GH secretion in women was more continuous,with pulses of similar amplitude throughout the 24 h.

Several methods were used to quantify the differences inthe pattern of GH secretion in men and women (Table I). As ameasure of uniformity of pulse amplitude, a SD for each sub-ject’s GH pulse amplitudes during the baseline study was cal-culated. GH pulse amplitudes were logarithmically trans-formed before analysis. This estimate of variation was greaterin men than in women (7.98 vs. 5.31

m

g/liter;

P

5

0.001). Asimilar analysis was performed after logarithmic transforma-tion of the 145 daily GH concentrations. A higher SD for daily

GH was found in men (5.22 vs. 4.03

m

g/liter;

P

5

0.04). Fig. 3shows a histogram of GH concentrations in these two groups.GH concentrations were either equal to or above the assay de-tection limit 98% of the time in men and 100% of the time inwomen. Although there was no difference between men andwomen in terms of absolute GH nadir, there was a differencein the frequency of biologically low GH concentrations. Reu-tens et al. (22) recently reported that subjects with severe or-ganic GH deficiency had virtually all plasma GH concentra-tions

,

0.5

m

g/liter during 24-h sampling. Using this value asan estimate of the minimum GH concentration for bioactivity,only 35

6

4% of the spontaneous plasma GH measurements inmen were above this value, whereas 53

6

6% of the concentra-tions in women exceeded this limit (

P

5

0.04).Deconvolution was used to determine differences in GH

secretion that would account for gender-specific GH concen-tration profiles. Fig. 4 (

top

) shows the BPF for men andwomen calculated as the deconvolution estimated pulse fre-

Figure 2. Composite picture of plasma GH con-centration profiles (mean6SE) in eight men (top) and eight women (bottom) during saline infusions.

M F

M F

M F

Time of Day (hr)

Hum

anMouse

Rat

Plas

ma

GH

(ng/

ml)

Pulsatile GH vs. Persistent GH Sex difference across species:Sustained GH-free

period in males(pulsatile GH)

vs. persistent GH stimulation in females

Inter-peak interval is key for sexually

dimorphic gene expression:

A minimum GH off-time is required for the male liver

gene expression profile

Liver sex differences emerge at pubertyat onset of strong pituitary GH secretion

Sex-specific steroid hydroxylase P450 mRNAs in rat liver

Male

Female

Female

Male

A. CYP2C11Male-specific Testosterone 16a-OHase

B. CYP2C12Female-specific steroid sulfate-OHase

+ IntermittentGH pulses

+ Persistent GHstimulation


By which mechanism(s) doesGH regulate sex differences

in liver metabolism?Fundamental biological question:

How does a cell distinguish a pulsatile vs. persistent input signal

Hypothesis: GH regulates Male and Female liver gene transcription by distinct intracellular signaling pathways

GH-Receptor complex

Tyrosinephosphorylation

via JAK2

STAT5GH Pulse-activated Transcription Factor (TF)

Enriched in male liver nuclei; Proposed mediator of male transcription

Waxman et al,J Biol Chem 1995

Anti-pY Western blotof rat liver nuclei

pY-STAT5

1 2 3 4

M M F F

STAT – Signal Transducer andActivator of Transcription

• STAT5 is activated directly, and repeatedly, by each male plasma GH pulse• GH pulses induce STAT5 Tyr-P, nuclear translocation, and DNA binding• Female GH pattern activates liver STAT5 persistently, at a lower level

STAT5 is activated by GH in a sex-dependent manner that

reflects the sex-dependent pattern of pituitary GH stimulation of

hepatocytes in the liver


Transcription factor (TF)• DNA sequence-specific binding protein that

activates transcription• >1,000 different TFs in the human genome• We can identify the specific sites in the genome

where each TF binds• Many TFs preferentially bind

at open chromatin regions

https://www.slideshare.net/avinashtiwari18/transcription-factor

PlasmaMembrane

PP Jak2

Jak2

GHR

GH

P

PP

P

P

P

STA

T5

SH2

mRNANucleusP

P

STAT

5

STAT5

pY-STAT5dimer

P

P

STA

T5

STAT

5

STAT5

PTPase

GH stimulates STAT5 Tyr-P---> nuclear translocation

TTCnnnGAA

Cytosol

Time (min)

STAT5bprotein

0’ 20’ 40’ 60’ 120’GH pulse

pY699-STAT5b

A. GH pulses stimulate STAT5-pY, translocation into the nucleus, then return back to the cytoplasm in the unphosphorylated state

B. Persistent GH activates a persistent, lower level of STAT5 signaling in the nucleus0 min 40 min 60 min20 min 120 min

Green: STAT5bRed: Nuclear DNA

Liver CWSV-1 cells in culture


ROLE of STAT5 in SEX-DEPENDENTLIVER GENE EXPRESSION

Lessons from Knockout Mouse Models

Loss of male-specific liver P450 geneexpression; loss of pubertal growth spurt in males, not females

STAT5b-KO

Human STAT5b-A630P exhibits GH insensitivity and body growth phenotype similar to male STAT5b-KO mouse Kofoed et al, NEJM

• 90% of all male-biased genes require STAT5b for expression in male liver

• 60% of female-biased genes are de-repressed in KO-male liver, indicating GH pulse/STAT5b strongly represses their expression in WT males

Udy et al, PNAS

Clodfelter et al, Mol Endo

HelenDavey

Where in the genome doesGH-activated STAT5 bind?

Do STAT5 binding sites differbetween male and female liver?

Key findings:Ø STAT5 binds to thousands of discrete genomic regions,

with binding enriched nearby sex-biased genesØ STAT5 shows sex-dependent binding at a subset of its

binding sitesØ STAT5 binds at open chromatin regions identified as DHS

DNase Hypersensitive Sites (DHS) identify genomic regulatory regions as accessible (open) chromatin sites

Mouse liver nuclei

ReleasedDNA

fragments

DHS DHSSequence, then map released

DNA fragments back to the genome

DNase

GenomicDNA

DNase Hypersensitive Site (DHS)

~400 nt

+ strand reads - strand readsnucleosome nucleosome


Female-biased DHS

Female gene

STAT5

STAT5

TTCN3GAA

Male-biased DHSMale gene

TTCN3GAA

Maleliver

Femaleliver

Maleliver

Femaleliver

Chromatin accessibility (DHS) is a major point of regulationKey mechanistic question: How do plasma GH patterns regulate these

sex-differences in chromatin accessibility ?

?

?

Pulsatile GH

Persistent GH

Two mechanisms explain male-bias of Male DHS

1. 70% of Male-DHS areopen constitutively in

male liver, and are closed in female liver; they are not directly affected by

each GH pulse

2. 30% of Male-DHS dynamically open and close

with each pulse ofGH-activated STAT5.

This chromatin opening does not occur in female liver.

STAT5 pulse-independent DHS

TTCN3GAA

TTCN3GAA

éê

STAT5 pulse-dependent DHS

TTCN3G

AA

TTCN3GAA

Andy RampersaudJeanette Connerney

GH

GH

GH

GH

time

time

Male liver

Male liver

Does STAT5 binding to dynamic,male-biased DHS inducepulsatile transcription

of male-biased liver genes?


GH/STAT5 pulse-induced chromatin opening can induce pulsatile gene transcription

Individual mouse livers

TTCN3G

AA

éê

TTCN3GAAPlas

ma

GH

puls

e

Time (h)

closeopen

Connerney et al (2017) Endocrinol

Endogenous plasma GH pulses induce pulsatile transcription of the male-specific Ces2b gene

STAT5 High STAT5 Low

Ces2b DHS

Rela

tive

DHS

Leve

l

Pulsatilechromatin opening

STAT5 High STAT5 Low

Ces2b hnRNARe

lativ

e le

vel (

qPCR

)

Pulsatile gene transcription rate

.

unsplicedprimary

transcript

GH STAT5 GH STAT5

Male geneexpression

Female geneexpression

Male liverchromatin state

Female liverchromatin state

Persistent GH

Pulsatile GH

CUX2BCL6

Sex bias in gene expression is enhanced by repressor TFs

GH Regulates Sex-specific Liver Chromatin States in a Dynamic Manner

GHcontin infusion

PlasmaGH

Why do some genes respond early (within hours), and

others very late (> 7 days), to a change in

plasma GH pattern?

Continuous GH overridesmale GH pulses, which:

represses male-biased genes induces female-biased genes

in male liver

Continuous GH

Underlying chromatin

environment

Changes in liver chromatin

state

Transcription factor binding

Feminize liver gene expression

+cGH


Histone H3 tail

Marmorstein (2001) Nat Rev Mol Cell Biol

(2) Heterochromatin

(1) Euchromatin

2. Compact, transcriptionally silent DNA; histone tail modifications enable nucleosomes to pack together.TFs cannot readily access DNA.

1. DNA is made accessible by histone acetylations à loosely packed nucleosomes. TFs can readily bind at open DNA and stimulate gene transcription.

Histonecore

BioRad

Histone ModificationsImpact on DNA Accessibility and Gene Responses

Nucleosome

Chromatin (Histone H3) Marks• K4-Me3, active promoters• K27-Me3, stable, inactive euchromatin• K4-Me1, K27ac, flank active, distal enhancers• K9-Me3, marks heterochromatin (gene silencing)• K36-Me3, marks transcribed genomic regions

Basal chromatin state in male liverdictates responsiveness to GHcont

Genes already in poised state or active staterespond more rapidly to a change in GH status

Active marks(H3K4me1, H3K27ac)Repressive marks(H3K27me3)

Fmo2, an early responding female-biased gene

Poised state

GHcontin

Active state

- repressive marks

Lau-Corona et al (2017) Mol Cell Biol

Cyp2b13, a late responding female-biased gene

Inactive state

- repressive marks

+ activatingmarks

GHcontin

Active state

GH STAT5 GH STAT5

Male geneexpression

Female geneexpression

Male liver cellchromatin state

Female liver cellchromatin state

Persistent GH

Pulsatile GH

CUX2BCL6

Sex Differences in the LiverMetabolism, liver disease and epigenetic mechanisms

Inactive state Active state

- repressive marks

+ activatingmarks

GHcontin

(2) Heterochromatin

(1) Euchromatin

2. Compact, transcriptionally silent DNA;

histone tail modifications enable

nucleosomes to pack together.

TFs cannot readily access DNA.

1. DNA is made accessible by

histone acetylations àloosely

packed nucleosomes. TFs can

readily bind at open DNA and stim

ulate gene transcription.

Histonecore

BioRad

STAT5

M/F

TTCN3G

AA

éê

TTCN3GAAPla

sma

GH

pul

se

Time (h)

closeopen

whitehead lecture 2/3/2020 © dj waxman 1wi.mit.edu/files/wi/cfile/whitehead_sexdiffs...

Documents