Mayor Bill de Blasio is expected to take part in the painting.
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The project was delayed a couple times and President Donald Trump tweeted that the "sign" would denigrate Fifth Avenue, calling it a "symbol of hate."
De Blasio responded in a tweet showing his support for the Black Lives Matter movement.
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"Here's what you don't understand: Black people BUILT 5th Ave and so much of this nation," the mayor said. "Your 'luxury' came from THEIR labor, for which they have never been justly compensated. We are honoring them. The fact that you see it as denigrating your street is the definition of racism."
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The mayor also said a majority of the NYPD are people of color.
As more murals pop up across New York City, a Black Lives Matter mural was unveiled in Harlem on Wednesday morning. The big, bold letters were drawn on Adam Clayton Powell Boulevard between 125th and 127th streets. It was painted by local artists and community group members.
De Blasio has pledged to create similar murals in each of the city's five boroughs.
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